TWI781700B - Method for producing immobilized virus particles and method for producing vaccine - Google Patents
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Abstract
本發明是關於一種疫苗,其包含固定化病毒粒子,其中,在放熱試驗中,以成為固定化病毒粒子的起源之病毒粒子或對應的去活化病毒粒子於3隻兔子中的放熱反應之和為基準計,固定化病毒粒子於3隻兔子中的放熱反應之和未達80%。 The present invention relates to a vaccine comprising immobilized virions, wherein, in the exothermic test, the sum of the exothermic reactions of the virions which are the origin of the immobilized virions or the corresponding deactivated virions in 3 rabbits is given by Based on the benchmark, the sum of the exothermic reactions of immobilized virus particles in 3 rabbits did not reach 80%.
Description
本發明是關於一種包含固定化病毒粒子之疫苗。更詳細而言,本發明是關於一種包含固定化病毒粒子之疫苗,其是藉由將病毒的粒子結構使用固定劑進行固定化,來抑制了副反應。 The present invention relates to a vaccine comprising immobilized virus particles. More specifically, the present invention relates to a vaccine comprising immobilized virus particles, which suppresses side effects by immobilizing the virus particle structure with a fixative.
流行性感冒病毒及日本腦炎病毒等之病毒所導致的感染症,依照病毒的種類、感染的對象,而有重症化的情況。作為對抗這種病毒所導致的感染症之防禦、預防的方法,已知有疫苗的接種等。 Infectious diseases caused by viruses such as influenza virus and Japanese encephalitis virus may become severe depending on the type of virus and the infected target. Vaccination and the like are known as methods of defense and prevention against infectious diseases caused by such viruses.
專利文獻1:國際公開第2016/010081號 Patent Document 1: International Publication No. 2016/010081
非專利文獻1:J.Infect.Dis.2009 200(6)841-848 非專利文獻2:Vaccine 2009 27(5)786-791Non-Patent Document 1: J.Infect.Dis.2009 200(6) 841-848 Non-Patent Document 2: Vaccine 2009 27 (5) 786-791
對抗流行性感冒病毒及日本腦炎病毒等的病毒之疫苗,有去活化疫苗與活毒疫苗的二種類在製造、市售著。其中,去活化疫苗大致區分為:將精製過的病毒粒子用福馬林等的去活化劑處理而製備的全顆粒疫苗(whole particle vaccine)、與將精製過的病毒粒子用有機溶劑或界面活性劑破壞(裂解化)而製備的裂解疫苗(split vaccine)。全顆粒疫苗是免疫原性高,在預防感染的功效方面較為優異。但是,全顆粒疫苗,會有出現較強局部反應及放熱反應(exothermic reaction)等的副反應的傾向。另一方面,裂解疫苗,由於減低了局部反應,也幾乎沒有放熱反應所以安全性上較優異。但是,在基礎免疫尚未成立的兒童或免疫反應已經衰弱的高齡者中,裂解疫苗的免疫原性有較低的傾向。因此,期望著開發出顯示比裂解疫苗更優異的功效(免疫原性),且安全性高的疫苗。 As vaccines against viruses such as influenza virus and Japanese encephalitis virus, two types of inactivated vaccines and live virus vaccines are manufactured and commercially available. Among them, deactivated vaccines are roughly divided into: whole particle vaccines prepared by treating purified virions with a deactivating agent such as formalin, and those prepared by treating purified virions with organic solvents or surfactants. A split vaccine prepared by destruction (lysing). Whole particle vaccines are highly immunogenic and have excellent efficacy in preventing infection. However, whole particle vaccines tend to have side effects such as strong local reactions and exothermic reactions. On the other hand, split vaccines are superior in safety because they reduce local reactions and have almost no exothermic reactions. However, the immunogenicity of split vaccines tends to be lower in children with pre-established basal immunizations or in older adults with weakened immune responses. Therefore, the development of a vaccine exhibiting superior efficacy (immunogenicity) and high safety compared to split vaccines has been desired.
本發明是鑑於上述事情而完成者,目的在於提供一種疫苗,其免疫原性高,且副反應受到抑制。The present invention was made in view of the above circumstances, and an object of the present invention is to provide a vaccine with high immunogenicity and suppressed side effects.
本案發明人為了解決上述問題而進行了深入研究的結果,令人驚訝的是發現到,藉由不破壞病毒粒子的粒子結構(裂解化)而利用固定化劑進行固定化的方式,來包含仍保有與本來的病毒粒子同等的成分及結構的病毒粒子(以下,有時稱為「固定化病毒粒子」)之疫苗,在免疫原性試驗中為同等,在放熱試驗等的副反應的成績中為優異,進而達到完成本發明。As a result of in-depth research conducted by the inventors of the present case to solve the above-mentioned problems, it was surprisingly found that by immobilizing with an immobilizing agent without destroying the particle structure (cracking) of the virus particles, the Vaccines that retain the same composition and structure as the original virus particles (hereinafter sometimes referred to as "immobilized virus particles") are equivalent in immunogenicity tests, and in the results of side effects such as exothermic tests For excellence, and then reach the completion of the present invention.
亦即,本發明提供以下的[1]~[15]。 [1]一種疫苗,其包含固定化病毒粒子, 其中,在放熱試驗中,以成為上述固定化病毒粒子的起源之病毒粒子或對應的去活化病毒粒子於3隻兔子中的放熱反應之和為基準計,上述固定化病毒粒子於3隻兔子中的放熱反應之和未達80%。 [2]如[1]所述之疫苗,其中,在放熱試驗中,上述固定化病毒粒子於3隻兔子中的放熱反應之和在1.3℃以下。 [3]一種疫苗,其包含固定化病毒粒子, 其中,以藉由成為上述固定化病毒粒子的起源之病毒粒子或對應的去活化病毒粒子而受到刺激的人類周邊血液單核細胞所產生的發炎性細胞激素的量為基準計,藉由上述固定化病毒粒子而受到刺激的人類周邊血液單核細胞所產生的發炎性細胞激素的量未達80%。 [4]如[1]~[3]中任一項所述之疫苗,其中,成為上述固定化病毒粒子的起源之病毒粒子包含:正黏液病毒粒子、黃病毒粒子、或小核糖核酸病毒粒子。 [5]如[4]所述之疫苗,其中,上述病毒粒子包含:流行性感冒病毒粒子、日本腦炎病毒粒子、或A型肝炎病毒粒子。 [6]如[5]所述之疫苗,其中,上述病毒粒子包含流行性感冒病毒粒子。 [7]如[6]所述之疫苗,其中,上述流行性感冒病毒粒子包含:A型流行性感冒病毒粒子、或B型流行性感冒病毒粒子。 [8]如[6]或[7]所述之疫苗,其中,上述流行性感冒病毒粒子包含被分類為:H1N1亞型株、H2N2亞型株、H3N2亞型株、H3N8亞型株、H5N1亞型株、H5N2亞型株、H5N6亞型株、H6N1亞型株、H7N3亞型株、H7N7亞型株、H7N9亞型株、H9N2亞型株、或H10N8亞型株的流行性感冒病毒粒子。 [9]如[5]所述之疫苗,其中,上述病毒粒子包含日本腦炎病毒粒子。 [10]如[9]所述之疫苗,其中,上述日本腦炎病毒粒子包含:北京-1株、中山株、SA14-14-2株、或P3株。 [11]如[1]~[10]中任一項所述之疫苗,其中,上述固定化病毒粒子表面的0%~90%的蛋白質沒有固定化。 [12]如[1]~[11]中任一項所述之疫苗,其中,相對於成為上述固定化病毒粒子的起源之病毒粒子的比活性也就是抗原含量/蛋白質含量,上述固定化病毒粒子的比活性的相對值在0%~95%。 [13]如[1]~[12]中任一項所述之疫苗,其中,上述固定化病毒粒子所具有的平均粒徑,是成為上述固定化病毒粒子的起源之病毒粒子或對應的去活化病毒粒子的粒徑的80%~150%。 [14]如[1]~[13]中任一項所述之疫苗,其中,當利用蔗糖密度梯度離心法來測定上述固定化病毒粒子時,會在蔗糖濃度35%以上檢測出峰。 [15]如[1]~[14]中任一項所述之疫苗,其中,當利用高效液相層析法來測定上述固定化病毒粒子時,可確認到單峰性的峰。 [16]如[1]~[15]中任一項所述之疫苗,其中,當對小鼠進行了免疫時,比起對上述固定化病毒粒子具特異性的IgG1,上述疫苗誘導出更多的對上述固定化病毒粒子具特異性的IgG2a。That is, the present invention provides the following [1] to [15]. [1] A vaccine comprising immobilized virus particles, Wherein, in the exothermic test, based on the sum of the exothermic reactions of the virion originating from the immobilized virion or the corresponding inactivated virion in 3 rabbits, the above-mentioned immobilized virion in 3 rabbits The sum of the exothermic reactions does not reach 80%. [2] The vaccine according to [1], wherein, in an exothermic test, the sum of exothermic reactions of the immobilized virus particles in three rabbits is 1.3°C or lower. [3] A vaccine comprising immobilized virus particles, Wherein, based on the amount of inflammatory cytokines produced by human peripheral blood mononuclear cells stimulated by the virion that is the source of the immobilized virion or the corresponding inactivated virion, the immobilized Human peripheral blood mononuclear cells stimulated with virions produced less than 80% of the inflammatory cytokines. [4] The vaccine according to any one of [1] to [3], wherein the virions that are the source of the immobilized virions include: orthomyxovirus particles, flavivirus particles, or picornavirus particles . [5] The vaccine according to [4], wherein the virions include influenza virions, Japanese encephalitis virions, or hepatitis A virions. [6] The vaccine according to [5], wherein the virus particles include influenza virus particles. [7] The vaccine according to [6], wherein the influenza virus particles include type A influenza virus particles or type B influenza virus particles. [8] The vaccine according to [6] or [7], wherein the influenza virus particles are classified into: H1N1 subtype strains, H2N2 subtype strains, H3N2 subtype strains, H3N8 subtype strains, H5N1 Influenza virions of subtype strain, H5N2 subtype strain, H5N6 subtype strain, H6N1 subtype strain, H7N3 subtype strain, H7N7 subtype strain, H7N9 subtype strain, H9N2 subtype strain, or H10N8 subtype strain . [9] The vaccine according to [5], wherein the virus particles include Japanese encephalitis virus particles. [10] The vaccine according to [9], wherein the Japanese encephalitis virus particles include Beijing-1 strain, Zhongshan strain, SA14-14-2 strain, or P3 strain. [11] The vaccine according to any one of [1] to [10], wherein 0% to 90% of the protein on the surface of the immobilized virion is not immobilized. [12] The vaccine according to any one of [1] to [11], wherein the immobilized virus is higher than the specific activity of the virion originating from the immobilized virion, that is, antigen content/protein content. The relative value of the specific activity of the particles is between 0% and 95%. [13] The vaccine according to any one of [1] to [12], wherein the average particle diameter of the immobilized virion is that of the virion originating from the immobilized virion or the corresponding derivation. 80% to 150% of the particle size of activated virus particles. [14] The vaccine according to any one of [1] to [13], wherein when the immobilized virus particles are measured by a sucrose density gradient centrifugation method, a peak is detected at a sucrose concentration of 35% or higher. [15] The vaccine according to any one of [1] to [14], wherein when the immobilized virus particles are measured by high performance liquid chromatography, a unimodal peak can be confirmed. [16] The vaccine according to any one of [1] to [15], wherein, when mice are immunized, the vaccine induces a higher level of expression than IgG1 specific for the immobilized virus particles. More IgG2a specific for the above-mentioned immobilized virions.
並且,本發明提供以下的[17]~[31]。 [17]一種固定化病毒粒子的製造方法,其包含一步驟,該步驟為在包含成為起源之病毒粒子或對應的去活化病毒粒子之懸浮液中添加固定劑。 [18]如[17]所述之製造方法,其中,上述固定劑包含醛類。 [19]如[18]所述之製造方法,其中,上述醛類選自由下列所組成之群組:甲醛、三聚甲醛、戊二醛、及該等之組合。Furthermore, the present invention provides the following [17] to [31]. [17] A method for producing immobilized virus particles, comprising a step of adding a fixative to a suspension containing originating virus particles or corresponding deactivated virus particles. [18] The production method according to [17], wherein the fixing agent contains aldehydes. [19] The production method according to [18], wherein the aldehyde is selected from the group consisting of formaldehyde, paraformaldehyde, glutaraldehyde, and combinations thereof.
〔20〕如〔19〕所述之製造方法,其中,上述醛類包含甲醛。 [20] The production method according to [19], wherein the aldehydes include formaldehyde.
〔21〕如〔20〕所述之製造方法,其中,以上述懸浮液和上述固定劑的總量為基準計,上述甲醛的濃度是0.005~0.5w/v%。 [21] The production method according to [20], wherein the concentration of the formaldehyde is 0.005 to 0.5 w/v% based on the total amount of the suspension and the fixative.
〔22〕如〔19〕所述之製造方法,其中,上述醛類包含戊二醛。 [22] The production method according to [19], wherein the aldehydes include glutaraldehyde.
〔23〕如〔22〕所述之製造方法,其中,以上述懸浮液和上述固定劑的總量為基準計,上述戊二醛的濃度是0.001~0.06w/v%。 [23] The production method according to [22], wherein the concentration of the glutaraldehyde is 0.001 to 0.06 w/v% based on the total amount of the suspension and the fixative.
〔24〕如〔17〕所述之製造方法,其中,上述固定劑包含碳二亞胺類。 [24] The production method according to [17], wherein the fixing agent contains carbodiimides.
〔25〕如〔24〕所述之製造方法,其中,上述碳二亞胺類選自由下列所組成之群組:二環己基碳二亞胺、二異丙基碳二亞胺、1-乙基-3-(3-二甲胺丙基)碳二亞胺鹽酸鹽、該等之類似物、及該等之組合。 [25] The production method according to [24], wherein the carbodiimides are selected from the group consisting of dicyclohexylcarbodiimide, diisopropylcarbodiimide, 1-ethylcarbodiimide, -3-(3-dimethylaminopropyl)carbodiimide hydrochloride, analogs thereof, and combinations thereof.
〔26〕如〔25〕所述之製造方法,其中,上述碳二亞胺類包含1-乙基-3-(3-二甲胺丙基)碳二亞胺鹽酸鹽。 [26] The production method according to [25], wherein the carbodiimides include 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride.
〔27〕如〔26〕所述之製造方法,其中,以上述懸浮液和上述固定劑的總量為基準計,上述1-乙基-3-(3-二甲胺丙基)碳二亞胺鹽酸鹽的濃度是0.05~1500mM。 [27] The production method according to [26], wherein the 1-ethyl-3-(3-dimethylaminopropyl)carbodiethylene The concentration of amine hydrochloride is 0.05~1500mM.
〔28〕如〔17〕~〔27〕中任一項所述之製造方法,其中,成為起源之上述病毒粒子,是使培養細胞、雞蛋或小鼠腦受到感染並回收而得的病毒粒子。 [28] The production method according to any one of [17] to [27], wherein the virus particle used as the origin is a virus particle obtained by infecting cultured cells, hen eggs, or mouse brain and collecting them.
[29]如[28]所述之製造方法,其中,上述培養細胞包含:初代細胞、或株化細胞。 [30]如[29]所述之製造方法,其中,上述培養細胞包含:Vero細胞(非洲綠猴腎細胞)、或MDCK(Madin-Darby Canine Kidney)細胞。 [31]一種疫苗的製造方法,其包含一步驟,該步驟為添加固定化病毒粒子,該固定化病毒粒子是藉由如[17]~[30]中任一項所述之製造方法而獲得的固定化病毒粒子。[29] The production method according to [28], wherein the cultured cells include primary cells or strained cells. [30] The production method according to [29], wherein the cultured cells include Vero cells (African green monkey kidney cells) or MDCK (Madin-Darby Canine Kidney) cells. [31] A method for producing a vaccine, comprising a step of adding immobilized virus particles obtained by the production method described in any one of [17] to [30] immobilized virus particles.
根據本發明,能夠提供一種疫苗,其免疫原性高,且副反應受到抑制。According to the present invention, it is possible to provide a vaccine with high immunogenicity and suppressed side effects.
以下,針對本發明之合適實施形態來詳細說明。但是,本發明並不限定於以下的實施形態。Hereinafter, suitable embodiments of the present invention will be described in detail. However, the present invention is not limited to the following embodiments.
[包含固定化病毒粒子之疫苗] 本實施形態之疫苗,為一種包含固定化病毒粒子之疫苗,在放熱試驗中,以成為上述固定化病毒粒子的起源之病毒粒子或對應的去活化病毒粒子於3隻兔子中的放熱反應之和為基準時,上述固定化病毒粒子於3隻兔子中的放熱反應之和(℃)是降低的。又,上述疫苗,以藉由成為上述固定化病毒粒子的起源之病毒粒子或對應的去活化病毒粒子而受到刺激的人類周邊血液單核細胞所產生的發炎性細胞激素的量為基準計,藉由固定化病毒粒子而受到刺激的人類周邊血液單核細胞所產生的發炎性細胞激素的量是降低的。亦即,包含固定化病毒粒子之疫苗的免疫原性,高於裂解疫苗的免疫原性,而局部反應及放熱反應等的副反應,則與裂解疫苗同等地受到抑制。在其他方面,與習知的包含去活化病毒粒子的全顆粒疫苗比較,固定化病毒粒子的穩定性較為優異。[Vaccines containing immobilized virus particles] The vaccine of this embodiment is a vaccine containing immobilized virus particles. In the exothermic test, the sum of the exothermic reactions of the virus particles that are the origin of the immobilized virus particles or the corresponding inactivated virus particles in three rabbits The sum (°C) of the exothermic reactions of the above-mentioned immobilized virus particles in 3 rabbits was reduced as a reference. In addition, the above-mentioned vaccine is based on the amount of inflammatory cytokines produced by human peripheral blood mononuclear cells stimulated by the virion that is the source of the above-mentioned immobilized virion or the corresponding deactivated virion, by The production of inflammatory cytokines by human peripheral blood mononuclear cells stimulated by immobilized virions was reduced. That is, the immunogenicity of the vaccine containing immobilized virions is higher than that of the split vaccine, and side effects such as local reactions and exothermic reactions are suppressed as much as the split vaccine. In other respects, the stability of immobilized virions is superior compared to conventional whole particle vaccines comprising deactivated virions.
所謂的「固定化病毒粒子」,是意味著沒有對宿主的感染能力,且藉由病毒粒子表面的蛋白質彼此交聯而使粒子結構被固定化的病毒粒子。固定化病毒粒子,由於維持著與原本的病毒粒子或對應的去活化病毒粒子同等的粒子性,故免疫原性高。固定化病毒粒子,是藉由利用固定劑處理成為起源之病毒粒子或對應的去活化病毒粒子而獲得。此處所謂的「固定劑」,是意味著藉由共價鍵將病毒粒子的蛋白質彼此交聯的藥劑。例如,固定劑是使表面抗原彼此、表面抗原與基質蛋白質或膜蛋白質,或者是基質蛋白質彼此或膜蛋白質彼此交聯,而保持病毒粒子的粒子結構的藥劑。The so-called "immobilized virus particle" means that it has no ability to infect the host, and the particle structure is immobilized by cross-linking the proteins on the surface of the virus particle. The immobilized virus particles maintain the same particle properties as the original virus particles or the corresponding inactivated virus particles, so the immunogenicity is high. Immobilized virions are obtained by treating the virions of origin or corresponding deactivated virions with a fixative. The term "fixing agent" here means an agent that cross-links the proteins of the virions with each other through covalent bonds. For example, a fixative is an agent that cross-links surface antigens, surface antigens and matrix proteins or membrane proteins, or matrix proteins or membrane proteins, thereby maintaining the particle structure of virions.
所謂的「去活化病毒粒子」,是意味著沒有對宿主的感染能力,且粒子結構沒有被固定化的病毒粒子。去活化病毒粒子,是藉由利用去活化劑處理成為起源之病毒粒子而獲得。流行性感冒病毒粒子的情況中,去活化病毒粒子,例如可以是藉由在包含流行性感冒病毒粒子的懸浮液中,以最終濃度成為0.02v/v%(以甲醛換算為0.0072~0.0076w/v%)的方式添加福馬林(36~38w/v%甲醛水溶液),並於4℃反應6週而獲得的去活化病毒粒子。日本腦炎病毒粒子的情況中,去活化病毒粒子,例如可以是市售的Vero細胞培養日本腦炎疫苗原料藥(化學及血清療法研究所製造,商品名「ENCEVAC」,包含利用0.08v/v%福馬林去活化完成的日本腦炎病毒粒子)。The so-called "deactivated virus particle" means a virus particle that has no ability to infect the host and the particle structure has not been immobilized. Inactivated virions are obtained by treating the originating virions with a deactivating agent. In the case of influenza virions, the deactivation of the virions can be achieved, for example, by making the final concentration 0.02v/v% (0.0072 to 0.0076w/ v%) by adding formalin (36-38w/v% formaldehyde aqueous solution) and reacting at 4°C for 6 weeks to obtain inactivated virus particles. In the case of Japanese encephalitis virions, the deactivated virions can be, for example, commercially available Vero cell cultured Japanese encephalitis vaccine raw materials (manufactured by the Institute of Chemistry and Serotherapy, trade name "ENCEVAC", including 0.08v/v % formalin deactivated Japanese encephalitis virions).
放熱試驗,是遵照在生物學性製劑基準(日本國,厚生勞動省告示第192號)所示之放熱試驗法的方法來進行。所謂的「放熱反應」,是意味著將檢體注射到耳靜脈內之後所測定而得的兔子的體溫(有時稱為「測定値」),與在注射前所測定而得的兔子的體溫(有時稱為「對照體溫」)的差(有時稱為「差體溫」)的最大値。此處,當差體溫為負値的時候,放熱反應是解釋為0。The exothermic test was carried out in accordance with the method of the exothermic test method shown in the biological agent standard (Japan, Ministry of Health, Labor and Welfare Notification No. 192). The so-called "exothermic reaction" means that the temperature of the rabbit measured after injecting the sample into the ear vein (sometimes called "measurement value") is different from the temperature of the rabbit measured before injection. (sometimes called "control temperature") difference (sometimes called "differential temperature"). Here, when the differential body temperature is negative, the exothermic reaction is interpreted as 0.
具體而言,是用以下的順序進行放熱試驗。首先,用生理食鹽水稀釋固定化病毒粒子,並將1mL中的蛋白質含量設成70μg(日本腦炎病毒粒子的情況)或設成240μg(流行性感冒病毒粒子的情況)的固定化病毒粒子當作試料。將上述試料,以每1kg體重為1~3mL的量接種到兔子,觀察發熱的上昇,直到6小時後為止。求出接種試料前的兔子的體溫(對照體溫)與接種後的兔子的體溫的差,將該差的最大値當作該兔子的放熱反應。對3隻兔子進行同樣的試驗,求出3隻兔子的放熱反應之和(℃)。Specifically, the exothermic test was performed in the following procedure. First, the immobilized virions were diluted with normal saline, and the protein content in 1 mL was set to 70 μg (in the case of Japanese encephalitis virions) or 240 μg (in the case of influenza virions). As a sample. The above-mentioned sample was inoculated into rabbits in an amount of 1 to 3 mL per 1 kg of body weight, and the rise in fever was observed until 6 hours later. The difference between the body temperature of the rabbit before inoculation with the sample (control body temperature) and the body temperature of the rabbit after inoculation was obtained, and the maximum value of the difference was regarded as the exothermic reaction of the rabbit. Carry out the same test on 3 rabbits, and calculate the sum (°C) of the exothermic reactions of the 3 rabbits.
上述疫苗,在放熱試驗中,以成為固定化病毒粒子的起源之病毒粒子或對應的去活化病毒粒子於3隻兔子中的放熱反應之和為基準計,固定化病毒粒子於3隻兔子中的放熱反應之和,亦可未達80%,亦可未達60%,亦可未達40%,亦可未達20%,亦可未達10%。下限雖然沒有特別限制,以成為固定化病毒粒子的起源之病毒粒子或對應的去活化病毒粒子於3隻兔子中的放熱反應之和為基準計,亦可在0%以上,亦可在20%以上。藉由將放熱反應之和設定在上述範圍內,而能夠提供一種疫苗,其與習知的包含去活化病毒粒子的全顆粒疫苗相比,副反應受到抑制。In the exothermic test of the above-mentioned vaccine, the exothermic reaction of the immobilized virus particles or the corresponding inactivated virus particles in three rabbits was used as the basis, and the rate of immobilized virus particles in three rabbits was The sum of exothermic reactions may be less than 80%, less than 60%, less than 40%, less than 20%, or less than 10%. Although the lower limit is not particularly limited, based on the sum of the exothermic reactions of the virion originating from the immobilized virion or the corresponding deactivated virion in 3 rabbits, it may be above 0% or 20%. above. By setting the sum of exothermic reactions within the above range, it is possible to provide a vaccine in which side effects are suppressed compared with conventional whole particle vaccines containing inactivated virions.
上述疫苗,在放熱試驗中,固定化病毒粒子於3隻兔子中的放熱反應之和,亦可在1.3℃以下,亦可在0.9℃以下,亦可在0.5℃以下。下限雖然沒有特別限制,亦可在0℃以上,亦可在0.6℃以上。藉由將放熱反應之和設定在上述範圍內,而能夠提供一種疫苗,其與習知的包含去活化病毒粒子的全顆粒疫苗相比,副反應受到抑制。For the above vaccine, in the exothermic test, the sum of the exothermic reactions of the immobilized virus particles in three rabbits may be below 1.3°C, below 0.9°C, or below 0.5°C. Although the lower limit is not particularly limited, it may be 0°C or higher, or 0.6°C or higher. By setting the sum of exothermic reactions within the above range, it is possible to provide a vaccine in which side effects are suppressed compared with conventional whole particle vaccines containing inactivated virions.
所謂的「發炎性細胞激素」,是伴隨發炎而產生的細胞激素的總稱,例如可舉出:IL-1β、IL-6、TNF-α、IFN-α、IFN-γ等。"Inflammatory cytokine" is a general term for cytokines produced along with inflammation, and examples thereof include IL-1β, IL-6, TNF-α, IFN-α, and IFN-γ.
所謂的「人類周邊血液單核細胞」(PBMC),是意味著由人類的周邊血液得到的淋巴球(包含T細胞、B細胞、NK細胞等)及單核球。The so-called "human peripheral blood mononuclear cells" (PBMC) means lymphocytes (including T cells, B cells, NK cells, etc.) and monocytes obtained from peripheral blood of humans.
發炎性細胞激素的量,是以遵照歐洲藥典MONOCYTE-ACTIVATION TEST(單核球活化試驗法)的方法,利用將藉由病毒粒子刺激人類周邊血液單核細胞(PBMC)時的發炎性細胞激素的產量進行定量來求得。作為上述方法,亦可為在歐洲藥典MONOCYTE-ACTIVATION TEST中進行了變更後述的實施例所示的測定條件的方法。The amount of inflammatory cytokines is based on the method of the European Pharmacopoeia MONOCYTE-ACTIVATION TEST (monocyte activation test method), using the inflammatory cytokines when human peripheral blood mononuclear cells (PBMC) are stimulated by virus particles Quantitative production is obtained. The method described above may be a method in which the measurement conditions shown in the Examples described later are changed in the European Pharmacopoeia MONOCYTE-ACTIVATION TEST.
上述疫苗,以藉由成為固定化病毒粒子的起源之病毒粒子或對應的去活化病毒粒子而受到刺激的人類周邊血液單核細胞所產生的發炎性細胞激素的量為基準計,藉由固定化病毒粒子而受到刺激的人類周邊血液單核細胞所產生的發炎性細胞激素的量亦可未達80%,亦可未達60%,亦可未達40%,亦可未達20%,亦可未達10%。下限雖然沒有特別限制,以藉由成為固定化病毒粒子的起源之病毒粒子或對應的去活化病毒粒子而受到刺激的人類周邊血液單核細胞所產生的發炎性細胞激素的量為基準計,亦可在0%以上,亦可在40%以上。透過將藉由固定化病毒粒子而受到刺激的人類周邊血液單核細胞所產生的發炎性細胞激素的量設定在上述範圍內,而能夠提供一種疫苗,其與習知的包含去活化病毒粒子的全顆粒疫苗相比,副反應受到抑制。The above-mentioned vaccine is based on the amount of inflammatory cytokines produced by human peripheral blood mononuclear cells stimulated by the virions which are the origin of the immobilized virions or the corresponding deactivated virions, by immobilizing The amount of inflammatory cytokines produced by human peripheral blood mononuclear cells stimulated by virus particles may also be less than 80%, or less than 60%, or less than 40%, or less than 20%, or less than 80%. May not reach 10%. Although the lower limit is not particularly limited, it is based on the amount of inflammatory cytokines produced by human peripheral blood mononuclear cells stimulated by the virion that is the source of the immobilized virion or the corresponding deactivated virion. It can be above 0% or above 40%. By setting the amount of inflammatory cytokines produced by human peripheral blood mononuclear cells stimulated by immobilized virions within the above-mentioned range, it is possible to provide a vaccine that is different from conventional vaccines containing deactivated virions. Compared with the whole particle vaccine, side effects were suppressed.
發炎性細胞激素為IL-1β的情況,藉由固定化病毒粒子而受到刺激的人類周邊血液單核細胞所產生的發炎性細胞激素之濃度,以包含上述人類周邊血液單核細胞的培養液為基準計,亦可在30pg/ml以下,亦可在20pg/ml以下。下限雖然沒有特別限制,但亦可在0pg/ml以上,亦可在5pg/ml以上。透過將藉由固定化病毒粒子而受到刺激的人類周邊血液單核細胞所產生的發炎性細胞激素之濃度設定在上述範圍內,而能夠提供一種疫苗,其與習知的包含去活化病毒粒子的全顆粒疫苗相比,副反應受到抑制。In the case where the inflammatory cytokine is IL-1β, the concentration of the inflammatory cytokine produced by human peripheral blood mononuclear cells stimulated by immobilized virus particles is set to be On a benchmark basis, it can also be below 30pg/ml or below 20pg/ml. Although the lower limit is not particularly limited, it may be 0 pg/ml or more, and may be 5 pg/ml or more. By setting the concentration of inflammatory cytokines produced by human peripheral blood mononuclear cells stimulated by immobilized virions within the above-mentioned range, it is possible to provide a vaccine that is different from conventional vaccines containing deactivated virions. Compared with the whole particle vaccine, side effects were suppressed.
發炎性細胞激素為IL-6的情況,藉由固定化病毒粒子而受到刺激的人類周邊血液單核細胞所產生的發炎性細胞激素之濃度,以包含上述人類周邊血液單核細胞的培養液為基準計,亦可在50pg/ml以下,亦可在40pg/ml以下。下限雖然沒有特別限制,但亦可在0pg/ml以上,亦可在5pg/ml以上。透過將藉由固定化病毒粒子而受到刺激的人類周邊血液單核細胞所產生的發炎性細胞激素之濃度設定在上述範圍內,而能夠提供一種疫苗,其與習知的包含去活化病毒粒子的全顆粒疫苗相比,副反應受到抑制。When the inflammatory cytokine is IL-6, the concentration of the inflammatory cytokine produced by the human peripheral blood mononuclear cells stimulated by the immobilized virus particles is set to On a benchmark basis, it can also be below 50pg/ml or below 40pg/ml. Although the lower limit is not particularly limited, it may be 0 pg/ml or more, and may be 5 pg/ml or more. By setting the concentration of inflammatory cytokines produced by human peripheral blood mononuclear cells stimulated by immobilized virions within the above-mentioned range, it is possible to provide a vaccine that is different from conventional vaccines containing deactivated virions. Compared with the whole particle vaccine, side effects were suppressed.
作為成為固定化病毒粒子的起源之病毒粒子,例如可舉出:痘病毒粒子、皰疹病毒粒子、正黏液病毒粒子、副黏液病毒粒子、棒狀病毒粒子、冠狀病毒粒子、沙狀病毒粒子、披衣病毒粒子、黃病毒粒子、崩芽病毒粒子、反轉錄病毒粒子、嗜肝DNA病毒粒子、腺病毒粒子、乳突狀瘤病毒粒子、乳突多瘤空泡病毒粒子、絲狀病毒粒子、里奧病毒粒子、小核糖核酸病毒粒子及杯狀病毒粒子。上述正黏液病毒粒子,例如可舉出流行性感冒病毒粒子。上述黃病毒粒子,例如可舉出日本腦炎病毒粒子。上述小核糖核酸病毒粒子,例如可舉出A型肝炎病毒粒子。Examples of virions that serve as the source of immobilized virions include: poxvirus particles, herpesvirus particles, orthomyxovirus particles, paramyxovirus particles, rhabdovirus particles, coronavirus particles, arenavirus particles, Clodden virion, flavivirion, collapse bud virion, retrovirion, hepadnavirion, adenovirion, papilloma virion, papillary polyoma vacuolar virion, filamentous virion, Rio virions, picorna virions, and calici virions. The above-mentioned orthomyxovirus particles include, for example, influenza virus particles. Examples of the above-mentioned flavivirus particles include Japanese encephalitis virus particles. Examples of the above-mentioned picornucleic acid virus particles include hepatitis A virus particles.
作為流行性感冒病毒粒子,例如可舉出A型流行性感冒病毒粒子和B型流行性感冒病毒粒子。在A型流行性感冒病毒粒子的情況中,例如可舉出被分類成H1N1亞型株、H2N2亞型株、H3N2亞型株、H3N8亞型株、H5N1亞型株、H5N2亞型株、H5N6亞型株、H6N1亞型株、H7N3亞型株、H7N7亞型株、H7N9亞型株、H9N2亞型株、或H10N8亞型株的流行性感冒粒子。Examples of influenza virions include type A influenza virions and type B influenza virions. In the case of type A influenza virus particles, for example, strains classified into H1N1 subtype, H2N2 subtype, H3N2 subtype, H3N8 subtype, H5N1 subtype, H5N2 subtype, H5N6 Influenza particles of subtype strain, H6N1 subtype strain, H7N3 subtype strain, H7N7 subtype strain, H7N9 subtype strain, H9N2 subtype strain, or H10N8 subtype strain.
作為日本腦炎病毒粒子,例如可舉出日本腦炎病毒粒子的北京-1株、中山株(中山-予研株)、或SA14-14-2株及P3株。Examples of Japanese encephalitis virus particles include Beijing-1 strain, Zhongshan strain (Zhongshan-Yoken strain), SA14-14-2 strain, and P3 strain of Japanese encephalitis virus particles.
固定化病毒粒子,由於如同裂解疫苗般粒子結構並未被破壞,故包含源自病毒粒子的基因組核酸(DNA、RNA等)。源自病毒粒子的基因組核酸可作為佐劑來作用。例如,在去活化小兒麻痺病毒疫苗中,有包含病毒基因組RNA的D抗原、與不含病毒基因組RNA的C抗原。C抗原的免疫原性弱,不會顯示出作為疫苗抗原的功效。具有作為疫苗抗原功效的分子物種只有D抗原。這件事暗示著,內含在疫苗中的病毒基因組RNA對於展現其功效是很重要的。因此,本實施形態之疫苗,可誘導Th1型反應。這與裂解流行性感冒疫苗是誘導Th2型反應的情況為互相對照。在小鼠中,比起藉由Th2型反應所誘導的IgG1亞型的抗體,藉由Th1型反應所誘導的IgG2a亞型的抗體對流行性感冒病毒的感染防禦能力更為優異。由此可見,能夠期待更加提升藉由上述疫苗的有效性。亦即,當對小鼠進行了免疫時,比起對固定化病毒粒子具特異性的IgG1,上述疫苗誘導出更多的對固定化病毒粒子具特異性的IgG2a。Immobilized virus particles contain genomic nucleic acid (DNA, RNA, etc.) derived from virus particles because the particle structure has not been destroyed like split vaccines. Genomic nucleic acid derived from virions can function as an adjuvant. For example, in an inactivated poliovirus vaccine, there is a D antigen that contains viral genomic RNA, and a C antigen that does not contain viral genomic RNA. The C antigen is weakly immunogenic and will not show efficacy as a vaccine antigen. The only molecular species that has efficacy as a vaccine antigen is the D antigen. This incident implies that the viral genomic RNA contained in the vaccine is important for its efficacy. Therefore, the vaccine of this embodiment can induce a Th1 type response. This is in contrast to the fact that the split influenza vaccine induces a Th2 type response. In mice, antibodies of the IgG2a subtype induced by a Th1-type response were superior in defense against influenza virus infection compared to antibodies of the IgG1 subtype induced by a Th2-type response. From this, it can be expected that the effectiveness of the above-mentioned vaccines will be further improved. That is, when mice were immunized, the vaccine induced more IgG2a specific for immobilized virions than IgG1 specific for immobilized virions.
固定化病毒粒子,當藉由蔗糖密度梯度離心法、高效液相層析法、及/或動態光散射法進行了測定時,亦可與原本的病毒粒子或對應的去活化病毒粒子為實質上相同的參數(例如:分子量、平均粒徑、密度、血球凝集素(HA)含量等)。Immobilized virions, as determined by sucrose density gradient centrifugation, high performance liquid chromatography, and/or dynamic light scattering, may also be substantially identical to native virions or corresponding deactivated virions. The same parameters (eg: molecular weight, average particle size, density, hemagglutinin (HA) content, etc.).
例如,固定化病毒粒子,亦可具有原本的病毒粒子或對應的去活化病毒粒子的粒徑的80%~150%的平均粒徑,亦可具有90%~140%的平均粒徑。日本腦炎病毒粒子的情況,固定化病毒粒子,亦可具有原本的病毒粒子或對應的去活化病毒粒子的粒徑的90%~130%的平均粒徑,亦可具有100%~120%的平均粒徑。For example, the immobilized virus particles may have an average particle diameter of 80% to 150% of that of the original virus particles or corresponding inactivated virus particles, or may have an average particle diameter of 90% to 140%. In the case of Japanese encephalitis virions, the immobilized virions may also have an average particle size of 90% to 130% of the particle size of the original virion or the corresponding inactivated virion, or may have an average particle size of 100% to 120%. The average particle size.
當固定化病毒粒子以流行性感冒病毒粒子作為起源時,固定化病毒粒子的平均粒徑,在以動態光散射法測定時是在150nm左右,亦可在120nm~180nm,亦可在130nm~170nm。在其他方面中,固定化病毒粒子當以流行性感冒病毒粒子作為起源時,固定化病毒粒子的平均粒徑,在以動態光散射法測定時亦可在100nm以上,亦可在120nm以上,亦可在130nm以上,亦可在150nm以上,亦可在170nm以上。上述平均粒徑,亦可在180nm以下,亦可在175nm以下,亦可在170nm以下。當固定化病毒粒子以日本腦炎病毒粒子作為起源時,固定化病毒粒子的平均粒徑,在以動態光散射法測定時是在90nm左右,亦可在80nm~110nm。在其他方面中,當固定化病毒粒子以日本腦炎病毒粒子作為起源時,固定化病毒粒子的平均粒徑,在以動態光散射法測定時亦可在70nm以上,亦可在80nm以上,亦可在90nm以上。上述平均粒徑,亦可在110nm以下,亦可在100nm以下。When the immobilized virus particles are derived from influenza virus particles, the average particle size of the immobilized virus particles is about 150nm when measured by dynamic light scattering method, or it can be between 120nm and 180nm, and it can also be between 130nm and 170nm . In other aspects, when the immobilized virus particles are derived from influenza virus particles, the average particle size of the immobilized virus particles can also be above 100nm, or above 120nm, or above 120nm when measured by the dynamic light scattering method. It can be above 130nm, or above 150nm, or above 170nm. The above average particle diameter may be 180 nm or less, 175 nm or less, or 170 nm or less. When the immobilized virus particles are derived from Japanese encephalitis virus particles, the average particle diameter of the immobilized virus particles is about 90 nm when measured by the dynamic light scattering method, and may also be 80 nm to 110 nm. In other aspects, when the immobilized virus particles are derived from Japanese encephalitis virus particles, the average particle size of the immobilized virus particles can also be more than 70nm, or more than 80nm, or more than 80nm when measured by dynamic light scattering method. Can be above 90nm. The above-mentioned average particle diameter may be 110 nm or less, or may be 100 nm or less.
當固定化病毒粒子以具有外膜的病毒粒子作為起源時,上述固定化病毒粒子的脂質成分的含量,相對於上述病毒粒子的脂質成分的含量,亦可同等。When the immobilized virion originates from a virion having an outer membrane, the content of the lipid component of the immobilized virion may be equal to the content of the lipid component of the virion.
固定化病毒粒子,當藉由蔗糖密度梯度離心法進行測定時,亦可在蔗糖濃度35%以上檢測出峰,亦可在蔗糖濃度45%以上且55%以下檢測出峰。上述蔗糖濃度,可藉由公知的方法來求得。例如,將包含固定化病毒粒子的檢體疊置於15~60%的蔗糖密度梯度,藉由在4℃、18000rpm(RCF=57500(×g))進行16小時的離心分離能夠求得上述蔗糖濃度。When the immobilized virus particles are measured by the sucrose density gradient centrifugation method, a peak can also be detected at a sucrose concentration of 35% or more, and a peak can also be detected at a sucrose concentration of 45% or more and 55% or less. The above-mentioned sucrose concentration can be obtained by a known method. For example, the above-mentioned sucrose can be obtained by superimposing a sample containing immobilized virus particles on a 15-60% sucrose density gradient, and performing centrifugation at 4°C, 18,000 rpm (RCF=57,500 (×g)) for 16 hours concentration.
固定化病毒粒子,當藉由高效液相層析(尺寸篩除層析(size exclusion chromatography;SEC))進行測定時,亦可確認到單峰性的峰。例如,使用尺寸篩除層析(商品名:TSKgel G6000PWXL(TOSOH公司製造)或Superose 6 10/300 GE(GE Healthcare公司製造))進行分子量測定時(溶析液:PBS,流速:0.5ml/min),亦可為溶出時間在14~15分鐘附近可確認單峰性的峰的日本腦炎病毒粒子的固定化病毒粒子,亦可為溶出時間在16~17分鐘附近可確認單峰性的峰的流行性感冒病毒粒子的固定化病毒粒子。The immobilized virus particles were also confirmed to have a unimodal peak when measured by high performance liquid chromatography (size exclusion chromatography (SEC)). For example, when measuring molecular weight using size exclusion chromatography (trade name: TSKgel G6000PWXL (manufactured by TOSOH) or
上述疫苗,其固定化病毒粒子表面的0%~90%的蛋白質,亦可沒有固定化,其固定化病毒粒子表面的5%~80%的蛋白質,亦可沒有固定化。The above-mentioned vaccine may or may not be immobilized with 0% to 90% of the protein on the surface of the virion, and may or may not be immobilized with 5% to 80% of the protein on the surface of the virion.
作為固定化病毒粒子表面的蛋白質,可舉出:表面抗原、基質蛋白質、膜蛋白質、及該等的組合。Examples of proteins immobilized on the surface of virions include surface antigens, matrix proteins, membrane proteins, and combinations thereof.
當病毒粒子為流行性感冒病毒粒子的情況,作為上述表面抗原,例如可舉出:血球凝集素(HA)、神經胺酸酶(NA)。作為上述基質蛋白質,例如可舉出M1蛋白質。作為上述膜蛋白質,例如可舉出M2蛋白質。When the virion is an influenza virion, examples of the surface antigen include hemagglutinin (HA) and neuraminidase (NA). Examples of the above-mentioned matrix protein include M1 protein. As said membrane protein, M2 protein is mentioned, for example.
當病毒粒子為日本腦炎病毒粒子的情況,作為上述表面抗原,例如可舉出:E蛋白質、M蛋白質。When the virions are Japanese encephalitis virions, examples of the surface antigens include E protein and M protein.
作為算出沒有固定化之蛋白質的比例的方法,例如,可舉出成為對象的蛋白質其原本的分子量中,求出固定化前與固定化後的蛋白質的量的變化率(殘存率)的方法。例如,可舉出一種算出M1蛋白質的殘存率(%)的方法,其是當固定化病毒粒子是將流行性感冒病毒粒子作為起源時,在進行了還原條件下的SDS-PAGE(聚丙烯醯胺膠體電泳)後,藉由密度測定法求出病毒的構成蛋白質之一的M1蛋白質的泳帶濃度,來算出M1蛋白質的殘存率(%)。這種情況,隨著提高固定劑的濃度,M1蛋白質的殘存率有降低的傾向。藉由應用此方法,能夠進行透過固定劑實行的交聯度分析。As a method of calculating the ratio of unimmobilized protein, for example, there is a method of calculating the rate of change (survival rate) in the amount of protein before and after immobilization from the original molecular weight of the target protein. For example, a method for calculating the remaining rate (%) of M1 protein can be given, which is to perform SDS-PAGE (polyacrylamide) under reducing conditions when the immobilized virus particles are derived from influenza virus particles. After amine colloid electrophoresis), the band concentration of the M1 protein, one of the constituent proteins of the virus, was obtained by densitometry, and the remaining rate (%) of the M1 protein was calculated. In this case, the remaining rate of M1 protein tends to decrease as the concentration of the fixative increases. By applying this method, analysis of the degree of cross-linking through a fixative can be performed.
於本實施形態中,當固定化病毒粒子是將流行性感冒病毒粒子作為起源時,M1蛋白質的殘存率亦可在5%~90%,M1蛋白質的殘存率亦可在10%~80%。換言之,M1蛋白質的5%~90%亦可沒有固定化,其10%~80%亦可沒有固定化。In this embodiment, when the immobilized virus particles are derived from influenza virus particles, the remaining rate of M1 protein may also be 5%-90%, and the remaining rate of M1 protein may also be 10%-80%. In other words, 5% to 90% of the M1 protein may not be immobilized, and 10% to 80% thereof may not be immobilized.
交聯度分析,亦能夠藉由求出固定化病毒粒子的比活性(抗原含量/蛋白質含量)來進行。使用於病毒粒子的抗原含量測定的單株抗體,會辨識中和抗原決定位,若中和抗原決定位的結構發生變化則比活性會降低。利用此性質,算出相對於沒有進行固定化的病毒粒子,固定化病毒粒子的比活性的相對値(%),評估交聯度。於本實施形態中,比活性的相對値在0~95%的範圍。當固定化病毒粒子是將日本腦炎病毒作為起源時,固定化日本腦炎病毒粒子的比活性的相對值亦可在90~95%,亦可在70~90%,亦可在50~70%。Cross-linking degree analysis can also be performed by obtaining the specific activity (antigen content/protein content) of immobilized virus particles. The monoclonal antibody used for the determination of the antigenic content of virions will recognize the neutralizing epitope, and if the structure of the neutralizing epitope changes, the specific activity will decrease. Using this property, the relative value (%) of the specific activity of immobilized virions relative to non-immobilized virions was calculated to evaluate the degree of cross-linking. In this embodiment, the relative value of the specific activity is in the range of 0 to 95%. When the immobilized virus particles are derived from Japanese encephalitis virus, the relative value of the specific activity of the immobilized Japanese encephalitis virus particles can also be 90-95%, or 70-90%, or 50-70%. %.
疫苗所包含的固定化病毒粒子的量,亦可依照病毒的種類或投予對象來適當選擇。例如,流行性感冒疫苗所包含的固定化病毒粒子的量(濃度),作為血球凝集素濃度,每一病毒株亦可為1~40μgHA/mL。The amount of immobilized virus particles contained in the vaccine can also be appropriately selected according to the type of virus or the subject of administration. For example, the amount (concentration) of the immobilized virus particles contained in the influenza vaccine may be 1 to 40 μg HA/mL as the hemagglutinin concentration per virus strain.
疫苗亦可為單一疫苗,其包含源自相同種類的病毒或細菌的抗原。疫苗亦可為混合疫苗,其包含源自複數種類的病毒或細菌的抗原。疫苗亦可為多價疫苗,其包含為同屬的病毒或細菌,但源自複數種類類型的抗原。例如,當疫苗為流行性感冒病毒疫苗的情況,亦可包含固定化A型流行性感冒病毒粒子或固定化B型流行性感冒病毒粒子中任一種,亦可該等兩者都包含。當疫苗為日本腦炎病毒疫苗的情況,亦可包含源自細胞培養或源自小鼠腦的病毒中任一種。上述流行性感冒病毒疫苗或日本腦炎病毒疫苗,亦可包含源自其他病毒或細菌的抗原。例如,亦可混合在白喉、百日咳、破傷風、去活化小兒麻痺病毒混合疫苗(DPT-IPV疫苗)中。A vaccine may also be a single vaccine comprising antigens derived from viruses or bacteria of the same species. A vaccine may also be a mixed vaccine comprising antigens derived from multiple species of viruses or bacteria. A vaccine may also be a multivalent vaccine, comprising antigens that are viruses or bacteria of the same genus, but originate from multiple types of species. For example, when the vaccine is an influenza virus vaccine, either one of immobilized influenza A virus particles or immobilized influenza B virus particles may be included, or both may be included. When the vaccine is a Japanese encephalitis virus vaccine, either of cell culture-derived or mouse brain-derived viruses may be contained. The above-mentioned influenza virus vaccine or Japanese encephalitis virus vaccine may also contain antigens derived from other viruses or bacteria. For example, it can also be mixed in diphtheria, pertussis, tetanus, inactivated poliovirus mixed vaccine (DPT-IPV vaccine).
疫苗的劑型,例如,亦可為液狀、粉末狀(冷凍乾燥粉末、乾燥粉末等)、膠囊狀、錠劑、冷凍狀態。The dosage form of the vaccine may be, for example, liquid, powder (lyophilized powder, dry powder, etc.), capsule, lozenge, or frozen state.
疫苗,亦可包含可容許作為醫藥品的載體。作為上述載體,能夠沒有限制地使用通常用於疫苗製造的載體。具體而言,上述載體可舉出:食鹽水、緩衝食鹽水、葡萄糖、水、甘油、等張水性緩衝液及該等的組合。疫苗亦可進一步適當調配乳化劑、保存劑(例如:硫柳汞)、等滲壓劑、pH調整劑、去活化劑(例如:福馬林)等。Vaccines may also contain pharmaceutically acceptable carriers. As the above-mentioned carrier, those generally used in vaccine production can be used without limitation. Specifically, examples of the carrier include saline, buffered saline, glucose, water, glycerin, isotonic aqueous buffer, and combinations thereof. The vaccine can also be further appropriately formulated with emulsifiers, preservatives (eg: thimerosal), isotonic agents, pH regulators, deactivating agents (eg: formalin), etc.
疫苗的投予途徑,例如亦可為:經皮投予、舌下投予、點眼投予、皮內投予、肌肉內投予、經口投予、經腸投予、經鼻投予、靜脈內投予、皮下投予、腹腔內投予、經口到肺的吸入投予。The administration route of the vaccine may be, for example, transdermal administration, sublingual administration, eyedrop administration, intradermal administration, intramuscular administration, oral administration, enteral administration, or nasal administration , intravenous administration, subcutaneous administration, intraperitoneal administration, and oral-to-pulmonary inhalation administration.
疫苗的投予方法,例如亦可為藉由注射器、經皮性貼劑、微針、可移植的緩釋性裝置、附有微針的注射器、無針裝置、或噴劑的投予方法。The vaccine may be administered by, for example, a syringe, transdermal patch, microneedle, implantable slow-release device, syringe with microneedle, needle-free device, or spray.
為了進一步提高免疫原性,疫苗亦可包含佐劑。作為佐劑,例如可舉出:鋁佐劑或包含鯊烯的水中油型乳化佐劑(AS03、MF59等);CpG和3-O-去醯基化-4’-單磷醯脂質A(3-O-desacyl-4'-monophosphoryl lipid A;MPL)等的類鐸受體(Toll-like receptor)的配位基;皂素系佐劑;聚γ-麩胺酸等的聚合物系佐劑;以及,幾丁聚糖和菊糖等的多醣類。To further increase immunogenicity, the vaccine may also contain adjuvants. Examples of adjuvants include: aluminum adjuvants or squalene-containing oil-in-water emulsified adjuvants (AS03, MF59, etc.); CpG and 3-O-deacylated-4'-monophosphoryl lipid A ( 3-O-desacyl-4'-monophosphoryl lipid A; MPL) and other Toll-like receptor ligands; saponin-based adjuvants; polymer-based adjuvants such as poly-γ-glutamic acid and polysaccharides such as chitosan and inulin.
作為成為對象的哺乳動物,例如可舉出:小鼠、大鼠、天竺鼠、倉鼠、兔子、貓、狗、綿羊、豬、牛、馬、山羊、猴、人等。本實施形態之疫苗,亦可對人使用,亦可對未滿5歳的兒童、65歳以上的高齡者使用。Examples of target mammals include mice, rats, guinea pigs, hamsters, rabbits, cats, dogs, sheep, pigs, cows, horses, goats, monkeys, and humans. The vaccine of this embodiment can also be used on humans, and it can also be used on children under 5 years old and elderly people over 65 years old.
疫苗雖然依照投予的目的、投予方法、投予對象的狀態(性別、年齡、體重、病況等)而異,但在流行性感冒疫苗對人投予的情況,亦可以每1次投予3.8μgHA~30μgHA的有效成分(固定化病毒粒子)的方式來使用。Although the vaccine varies depending on the purpose of administration, the method of administration, and the state of the recipient (sex, age, weight, disease condition, etc.), in the case of administering influenza vaccine to humans, it can also be administered every time 3.8 μg HA ~ 30 μg HA active ingredient (immobilized virus particles) is used.
[固定化病毒粒子的製造方法] 本實施形態之固定化病毒粒子的製造方法,其包含一步驟,該步驟為對成為起源之病毒粒子或對應的去活化病毒粒子,將粒子結構進行固定化。[Method for producing immobilized virus particles] The method for producing immobilized virus particles according to this embodiment includes a step of immobilizing the particle structure of the original virus particle or the corresponding inactivated virus particle.
作為提高疫苗安全性的技術,從習知可知,使用界面活性劑或有機溶劑來破壞具有外膜的病毒粒子(裂解化)的方法。然而,在這些方法,隨著粒子的崩壞而疫苗的有效性(免疫原性)有降低的傾向。在上述製造方法,由於是將成為起源之病毒粒子或對應的去活化病毒粒子,利用會引起與病毒粒子蛋白質的共價鍵的固定劑來處理,故病毒粒子結構能夠維持,就結果而言,變得能維持疫苗的有效性,而且提高安全性。As a technique for improving the safety of vaccines, a method of destroying (cracking) virions having an outer membrane using a surfactant or an organic solvent is conventionally known. However, in these methods, the effectiveness (immunogenicity) of the vaccine tends to decrease as the particles collapse. In the above-mentioned production method, since the original virion or the corresponding deactivated virion is treated with a fixative that causes a covalent bond with the virion protein, the structure of the virion can be maintained. As a result, It becomes possible to maintain effectiveness of vaccine, and to improve safety.
上述製造方法中,亦可進一步包含下列步驟:培養宿主的步驟、使宿主感染病毒的步驟、使病毒在宿主內增殖的步驟、從宿主回收病毒粒子的步驟、或將回收後的病毒粒子進行去活化的步驟。In the above production method, the following steps may be further included: a step of culturing the host, a step of infecting the host with a virus, a step of multiplying the virus in the host, a step of recovering the virus particles from the host, or removing the recovered virus particles. Activation steps.
病毒粒子,亦可為於宿主感染病毒粒子,使之增殖後,從宿主回收後的病毒粒子。宿主亦可依照病毒粒子的種類來適當選擇。將病毒粒子進行去活化的方法,使用公知的方法即可,例如可舉出利用福馬林等的去活化劑來進行去活化的方法。當病毒粒子為流行性感冒病毒粒子的情況,作為宿主,例如可舉出:培養細胞、雞蛋及小鼠腦。作為培養細胞,可為初代細胞亦可為株化細胞。作為培養細胞,例如可舉出Vero細胞及MDCK細胞。 Virus particles may be virus particles that are recovered from the host after the host is infected with the virus particles and allowed to proliferate. The host can also be appropriately selected according to the type of virus particles. As a method of deactivating virions, known methods may be used, for example, a method of deactivating using a deactivating agent such as formalin. When the virion is an influenza virion, examples of the host include cultured cells, eggs, and mouse brain. As cultured cells, primary cells or strained cells may be used. Examples of cultured cells include Vero cells and MDCK cells.
作為流行性感冒病毒的感染、增殖方法,對本發明所屬技術領域中具有通常知識者而言,有下述公知的方法:將雞蛋或Vero細胞作為宿主來使用的方法(Vaccine.1998May-Jun;16(9-10):960-8.)、將Vero細胞作為宿主來使用的方法(Vaccine.2007August10;25(32):6028-6036.)、將MDCK細胞作為宿主來使用的方法(JVirol.2012Nov;86(22):12341-50)。 As a method of infection and propagation of influenza virus, those who have ordinary knowledge in the technical field of the present invention have the following known methods: the method of using egg or Vero cells as a host (Vaccine. 1998 May-Jun; 16 (9-10): 960-8.), a method using Vero cells as a host (Vaccine.2007August10; 25(32): 6028-6036.), a method using MDCK cells as a host (JVirol.2012Nov ;86(22):12341-50).
病毒粒子的固定化 Immobilization of virions
上述進行固定化的步驟,可例示利用固定劑來處理成為起源之病毒粒子或對應的去活化病毒粒子的方法,例如可舉出在包含成為起源之病毒粒子或對應的去活化病毒粒子的懸浮液中添加固定劑的步驟。懸浮液中的病毒粒子的濃度,亦可依照病毒的種類、固定劑的種類及其濃度等來適當變更。例如,懸浮液中的病毒粒子的濃度,作為病毒粒子的蛋白質的濃度,亦可在60~90μg/mL,亦可在300~3000μg/mL,亦可在500~2500μg/mL。 The above-mentioned immobilization step can be exemplified by a method of treating the original virion or the corresponding inactivated virion with a fixative, for example, in a suspension containing the original virion or the corresponding inactivated virion In the step of adding fixative. The concentration of virus particles in the suspension can also be appropriately changed according to the type of virus, the type and concentration of the fixative, and the like. For example, the concentration of virus particles in the suspension may be 60 to 90 μg/mL, 300 to 3000 μg/mL, or 500 to 2500 μg/mL as the concentration of protein of the virus particles.
固定劑的種類,能夠依照病毒的種類適當變更。例如,上述固定劑可舉出:有機溶劑、醛類、二亞胺酯、雙(3,5-二溴水楊基)延胡索酸酯(Bis(3,5-dibromosalicyl)fumarate;DBBF)、碳二亞胺類、及該等的組合。作為有機溶劑,例如可舉出: 甲醇、乙醇、丙酮、及該等的組合。作為醛類,例如可舉出:甲醛(FA)(例如,福馬林)、三聚甲醛、戊二醛(GA)、及該等的組合。作為碳二亞胺類,例如可舉出:二環己基碳二亞胺(DCC)、二異丙基碳二亞胺(DIC)、1-乙基-3-(3-二甲胺丙基)碳二亞胺鹽酸鹽(EDC)、該等的類似物及該等的組合。 The type of fixative can be appropriately changed according to the type of virus. For example, the aforementioned fixatives include: organic solvents, aldehydes, diimide esters, bis(3,5-dibromosalicyl)fumarate (Bis(3,5-dibromosalicyl)fumarate; DBBF), carbon diacetate imines, and combinations thereof. Examples of organic solvents include: Methanol, ethanol, acetone, and combinations thereof. Examples of aldehydes include formaldehyde (FA) (for example, formalin), paraformaldehyde, glutaraldehyde (GA), and combinations thereof. Examples of carbodiimides include: dicyclohexylcarbodiimide (DCC), diisopropylcarbodiimide (DIC), 1-ethyl-3-(3-dimethylaminopropyl ) carbodiimide hydrochloride (EDC), analogs thereof and combinations thereof.
固定劑的濃度,亦可依照病毒的種類、固定劑的種類適當變更。當固定劑包含甲醛的情況,甲醛的濃度以包含病毒粒子的懸浮液及固定劑的總量為基準計,亦可在0.005~0.5w/v%。當上述甲醛的濃度未達0.005w/v%時,固定化會變弱,有著粒子結構難以保持的傾向。當上述甲醛的濃度超過0.5w/v%時,固定化強,有著藉由交聯的化學修飾過度進行的傾向。從要更加提升HA活性的觀點而言,以上述懸浮液及固定劑的總量為基準計,甲醛的濃度亦可在0.01~0.5w/v%,亦可在0.018~0.152w/v%,亦可在0.029~0.152w/v%,亦可在0.029~0.076w/v%。使用包含甲醛的固定劑的方法,亦可用在病毒粒子為流行性感冒病毒粒子、日本腦炎病毒粒子的情況。 The concentration of the fixative can also be appropriately changed according to the type of virus and the type of fixative. When the fixative contains formaldehyde, the concentration of formaldehyde may be 0.005-0.5w/v% based on the total amount of the suspension containing virus particles and the fixative. When the concentration of the above-mentioned formaldehyde is less than 0.005w/v%, the immobilization becomes weak, and the particle structure tends to be difficult to maintain. When the concentration of the above-mentioned formaldehyde exceeds 0.5 w/v%, the immobilization is strong, and the chemical modification by cross-linking tends to proceed excessively. From the point of view of further enhancing the activity of HA, the concentration of formaldehyde can also be 0.01~0.5w/v%, or 0.018~0.152w/v%, based on the total amount of the above-mentioned suspension and fixative. It can also be 0.029~0.152w/v%, or 0.029~0.076w/v%. The method of using a fixative containing formaldehyde can also be used when the virions are influenza virions or Japanese encephalitis virions.
當固定劑為福馬林(36~38w/v%甲醛水溶液)時,福馬林濃度以上述懸浮液及固定劑的總量為基準計,亦可在0.014~0.4v/v%,亦可在0.05~0.4v/v%,亦可在0.08~0.4v/v%,亦可在0.08~0.2v/v%。When the fixative is formalin (36~38w/v% formaldehyde aqueous solution), the formalin concentration can also be 0.014~0.4v/v% or 0.05% based on the total amount of the above-mentioned suspension and fixative. ~0.4v/v%, or 0.08~0.4v/v%, or 0.08~0.2v/v%.
當固定劑包含戊二醛時,以上述懸浮液及固定劑的總量為基準計,戊二醛的濃度亦可在0.001~0.06w/v%,亦可在0.002~0.05w/v%,亦可在0.004~0.02w/v%,亦可在0.005~0.01w/v%。當上述濃度未達0.001w/v%時,使用了日本腦炎病毒粒子作為病毒粒子時,粒子有凝集的傾向。當上述濃度超過0.06w/v%時,使用了日本腦炎病毒粒子作為病毒粒子時,主要結構蛋白質也就是E蛋白質的抗原決定位有失活的傾向。使用戊二醛作為固定劑的方法,亦可用在病毒粒子為流行性感冒病毒粒子、日本腦炎病毒粒子的情況。When the fixative contains glutaraldehyde, the concentration of glutaraldehyde can also be 0.001-0.06w/v%, or 0.002-0.05w/v%, based on the total amount of the above-mentioned suspension and fixative, It can also be 0.004~0.02w/v%, or 0.005~0.01w/v%. When the above-mentioned concentration is less than 0.001 w/v%, when Japanese encephalitis virus particles are used as the virus particles, the particles tend to aggregate. When the above-mentioned concentration exceeds 0.06w/v%, when Japanese encephalitis virus particles are used as the virus particles, the epitope of E protein, which is the main structural protein, tends to be inactivated. The method of using glutaraldehyde as a fixative can also be used when the virions are influenza virions or Japanese encephalitis virions.
當固定劑包含EDC時,以上述懸浮液及固定劑的總量為基準計,EDC的濃度亦可在0.05~1500mM,亦可在0.15~500mM,亦可在5~50mM。使用包含EDC的固定劑的方法,亦可用在病毒粒子為流行性感冒病毒粒子、日本腦炎病毒粒子的情況。When the fixative includes EDC, based on the total amount of the above-mentioned suspension and fixative, the concentration of EDC can also be 0.05-1500mM, 0.15-500mM, or 5-50mM. The method using a fixative containing EDC can also be used when the virions are influenza virions or Japanese encephalitis virions.
利用固定劑處理時的溫度,亦可依照病毒的種類、固定劑的種類、固定劑的濃度等來適當變更。上述溫度亦可在0℃(冰浴)~37℃,亦可在4℃~37℃,亦可在25℃~37℃。The temperature during the treatment with the fixative can also be appropriately changed according to the type of virus, the type of fixative, the concentration of the fixative, and the like. The above temperature can also be 0°C (ice bath) to 37°C, 4°C to 37°C, or 25°C to 37°C.
利用固定劑處理時的期間(處理時間),亦可依照病毒的種類、固定劑的種類、固定劑的濃度、處理的溫度等來適當變更。上述期間亦可在1日到4週之間,亦可在3日到4週之間,亦可在1週到4週之間。當使用EDC作為固定劑的情況,上述期間亦可在5分鐘~24小時,亦可在0.5小時~24小時,亦可在2小時~20小時。The period of treatment with a fixative (treatment time) can also be appropriately changed according to the type of virus, the type of fixative, the concentration of the fixative, the temperature of the treatment, and the like. The above period may also be between 1 day and 4 weeks, or between 3 days and 4 weeks, or between 1 week and 4 weeks. When EDC is used as a fixative, the above-mentioned period may be 5 minutes to 24 hours, or 0.5 hours to 24 hours, or 2 hours to 20 hours.
為了停止藉由固定劑的交聯的進行,亦可使用甘胺酸等的胺基酸來進行阻停處理(quenching treatment)。另外,阻停處理有著以疫苗的穩定性、免疫原性、及安全性的提升為目的而進行的情況。In order to stop the progress of the cross-linking by the fixative, it is also possible to perform quenching treatment using amino acids such as glycine. In addition, the blocking treatment is sometimes performed for the purpose of improving the stability, immunogenicity, and safety of the vaccine.
視需要,亦可進一步包含將經回收的固定化病毒粒子進行精製的步驟。作為精製固定化病毒粒子的方法,雖然可藉由公知的方法適當進行,例如可舉出使用超過濾膜來過濾的方法。If necessary, a step of refining the recovered immobilized virus particles may be further included. As a method of purifying immobilized virus particles, known methods can be suitably used, for example, a method of filtration using an ultrafiltration membrane is mentioned.
本實施形態之疫苗的製造方法,包含一步驟,該步驟是添加藉由上述固定化病毒粒子的製造方法所獲得的固定化病毒粒子。在疫苗的製造方法中,亦可進一步包含一步驟,該步驟是添加可容許作為醫藥品的載體、乳化劑、保存劑、等滲壓劑、pH調整劑、去活化劑等。The method for producing a vaccine according to this embodiment includes a step of adding the immobilized virus particles obtained by the above-mentioned method for producing immobilized virus particles. The vaccine production method may further include a step of adding a pharmaceutically acceptable carrier, emulsifier, preservative, isotonic agent, pH adjuster, deactivator, and the like.
在疫苗接種上,較佳是能夠給予對象與實際感染時同樣的免疫的質與量,相對於藉由實際感染所引起的免疫,藉由疫苗所誘導的免疫的仿效性來決定其功效。在疫苗中,亦可包含全部的作為用以防禦感染的抗原的病毒蛋白質。本案發明人認為,若是根據用以提高病毒蛋白質的免疫原性的源自病毒的基因組核酸的存在、病毒粒子的大小、形狀等,各自分別在免疫反應中起作用,則所謂最佳的疫苗就是與實際的病毒具有較為相近的成分及結構者。本實施形態之固定化病毒粒子,藉由僅利用固定劑進行固定化,除此以外由於具有與原本的病毒粒子同等的成分及結構,而能提供一種疫苗,其免疫原性高,且副反應受到抑制。 [實施例]In terms of vaccination, it is preferable to be able to give the subject the same quality and quantity of immunity as the actual infection. Compared with the immunity caused by the actual infection, the efficacy is determined by the imitation of the immunity induced by the vaccine. In vaccines, all viral proteins can also be included as antigens for defense against infection. The inventors of the present case believe that if each plays a role in the immune response based on the presence of genomic nucleic acid derived from the virus, the size and shape of the virus particle, etc., which are used to increase the immunogenicity of the virus protein, the so-called optimal vaccine is Those that have relatively similar components and structures to actual viruses. The immobilized virions of this embodiment can provide a vaccine with high immunogenicity and no side effects due to immobilization only with a fixative, and having the same composition and structure as the original virions. suppressed. [Example]
以下,藉由實施例來詳細說明本發明,但本發明並不限定於這些實施例。Hereinafter, the present invention will be described in detail with examples, but the present invention is not limited to these examples.
[實施例1] 1.源自流行性感冒病毒粒子的抗原的製備 (1)FA固定化流行性感冒病毒粒子的製備 甲醛(FA)處理 將A型流行性感冒病毒H1N1亞型株(A/California/07/2009(X-179A)株,以下有時記載為「A/CA株」)接種到11日齡的發育雞蛋的絨毛膜尿囊腔內,於34℃培養了2日。將獲得的絨毛膜尿囊液經過澄清化後,藉由超離心分離使流行性感冒病毒粒子沈澱。將上述流行性感冒病毒粒子利用磷酸緩衝生理食鹽水(PBS)使之再次懸浮而獲得懸浮液。將獲得的懸浮液利用蔗糖密度梯度離心法(RCF=57500(×g),16小時)進行離心分離,並藉由回收蔗糖濃度為33%~50%的級分(fraction),精製成流行性感冒粒子。以精製後的流行性感冒病毒粒子的蛋白質最終濃度成為500μg/mL的方式,稀釋獲得的級分,獲得了懸浮液。之後,以最終濃度成為0.05~0.20v/v%(用甲醛換算為0.018~0.076w/v%)的方式添加福馬林(36~38w/v%甲醛水溶液)到上述懸浮液中,於25℃使之反應1週。反應結束後,利用PBS來透析反應液,藉由去除甲醛獲得了固定化流行性感冒病毒粒子(以下有時記載為「FA固定化流行性感冒病毒粒子」)。[Example 1] 1. Preparation of Antigens Derived from Influenza Virions (1) Preparation of FA-immobilized influenza virus particles Formaldehyde (FA) treatment The H1N1 subtype strain of influenza A virus (A/California/07/2009 (X-179A) strain, hereinafter sometimes referred to as "A/CA strain") was inoculated into the chorionic urine of 11-day-old developing eggs. In the cyst cavity, cultured at 34°C for 2 days. After clarification of the obtained chorioallantoic fluid, influenza virus particles were precipitated by ultracentrifugation. The above-mentioned influenza virus particles were resuspended in phosphate-buffered saline (PBS) to obtain a suspension. The obtained suspension was centrifuged by sucrose density gradient centrifugation (RCF=57500 (×g), 16 hours), and the fraction (fraction) with a sucrose concentration of 33% to 50% was recovered and refined into a popular cold particles. The obtained fraction was diluted so that the final protein concentration of the purified influenza virions became 500 μg/mL to obtain a suspension. After that, add formalin (36-38w/v% formaldehyde aqueous solution) to the above suspension so that the final concentration becomes 0.05-0.20v/v% (0.018-0.076w/v% in terms of formaldehyde) Let it react for 1 week. After the reaction, the reaction solution was dialyzed with PBS to obtain immobilized influenza virus particles (hereinafter sometimes referred to as "FA immobilized influenza virus particles") by removing formaldehyde.
(2)去活化流行性感冒病毒粒子的製備 上述懸浮液中,以最終濃度成為0.02v/v%(用甲醛換算為0.0072~0.0076w/v%)的方式添加福馬林(36~38w/v%甲醛水溶液),於4℃使之反應6週~8週。反應結束後,利用PBS來透析反應液,藉由去除甲醛獲得了去活化流行性感冒病毒粒子。利用同樣的方法,亦針對其他的流行性感冒病毒株(亞型株)製備去活化流行性感冒病毒粒子,在實施例1~6中作為比較對照來使用。(2) Preparation of deactivated influenza virus particles Add formalin (36-38w/v% formaldehyde aqueous solution) to the above suspension so that the final concentration becomes 0.02v/v% (0.0072-0.0076w/v% in terms of formaldehyde), and react at 4°C6 Week to 8 weeks. After the reaction, the reaction solution was dialyzed with PBS to obtain deactivated influenza virus particles by removing formaldehyde. Using the same method, deactivated influenza virus particles were also prepared for other influenza virus strains (subtype strains), which were used as comparative controls in Examples 1-6.
(3)裂解流行性感冒病毒抗原的製備 作為比較對照的裂解流行性感冒病毒抗原(以下有時記載為「裂解流感抗原」),是使用了流行性感冒HA疫苗(化學及血清療法研究所製造,商品名「流行性感冒HA疫苗“化血研”」)所包含的各株的原液。(3) Preparation of Split Influenza Virus Antigen As a comparative control split influenza virus antigen (hereinafter sometimes referred to as "split influenza antigen"), an influenza HA vaccine (manufactured by the Institute of Chemical and Serotherapy, trade name "Influenza HA Vaccine" Blood research "") stock solution of each strain contained.
2.放熱試驗 遵照生物學性製劑基準(日本國,厚生勞動省告示第192號),進行了放熱試驗。將去活化流行性感冒病毒粒子、FA固定化流行性感冒病毒粒子及裂解流感抗原利用生理食鹽水來稀釋,將1mL中的蛋白質含量達240μg者作為試料。將上述試料以每1kg體重為1~3mL的量接種到兔子,觀察發熱的上昇,直到6小時後為止。求出接種試料前的兔子體溫(對照體溫)與接種後的兔子體溫的差,將該差的最大値作為該兔子的放熱反應。對3隻兔子進行了同樣的試驗。3隻兔子的放熱反應之和(℃)顯示於表1。2. Exothermic test Exothermic tests were performed in accordance with biological preparation standards (Notice No. 192 of the Ministry of Health, Labor and Welfare, Japan). Deactivated influenza virions, FA-immobilized influenza virions, and split influenza antigens were diluted with saline, and those with a protein content of 240 μg in 1 mL were used as samples. The above-mentioned sample was inoculated into rabbits in an amount of 1 to 3 mL per 1 kg of body weight, and the rise in fever was observed until 6 hours later. The difference between the body temperature of the rabbit before inoculation with the sample (control body temperature) and the body temperature of the rabbit after inoculation was obtained, and the maximum value of the difference was regarded as the exothermic reaction of the rabbit. The same experiment was carried out on 3 rabbits. The sum (°C) of the exothermic responses of the 3 rabbits is shown in Table 1.
[表1] [Table 1]
FA固定化流行性感冒病毒粒子,都沒有確認到1.3℃以上的放熱反應之和,與去活化流行性感冒病毒粒子相比,可見到3℃以上的放熱反應之和的減少。由結果可知,FA固定化流行性感冒病毒粒子與裂解流感抗原同樣,放熱反應之和相當低。由此亦教示了,FA固定化流行性感冒病毒粒子,與裂解流感抗原同樣地具有高安全性。The sum of exothermic reactions above 1.3°C was not confirmed in any of the FA-immobilized influenza virions, and the sum of exothermic reactions above 3°C was reduced compared with the deactivated influenza virions. It can be seen from the results that the sum of the exothermic reactions of FA-immobilized influenza virus particles is the same as that of splitting influenza antigens. This also teaches that FA-immobilized influenza virions are as highly safe as split influenza antigens.
3.定量發炎性細胞激素的產量 以遵照歐洲藥典MONOCYTE-ACTIVATION TEST的方法,來定量發炎性細胞激素(IL-6)的產量,該發炎性細胞激素是藉由利用了濃度為0.05v/v%、0.08v/v%、0.11v/v%的福馬林固定而成的FA固定化流行性感冒病毒粒子或去活化流行性感冒病毒粒子來刺激人類周邊血液單核細胞(PBMC)時的發炎性細胞激素。具體而言,歐洲藥典MONOCYTE-ACTIVATION TEST中的將人類PBMC以最少4位供給者份作為混合池來使用的部份,變更成1位供給者份,而進行了測定。FA固定化流行性感冒病毒粒子及去活化流行性感冒病毒粒子的細胞激素的產量的結果顯示於表2。可知FA固定化流行性感冒病毒粒子中的IL-6的產量,與去活化流行性感冒病毒粒子相比相當低。由此教示了,FA固定化流行性感冒病毒粒子,與去活化流行性感冒病毒粒子相比具有高安全性。3. Quantification of Inflammatory Cytokines Production Quantify the production of inflammatory cytokines (IL-6) by following the method of the European Pharmacopoeia MONOCYTE-ACTIVATION TEST. Inflammatory cytokines in human peripheral blood mononuclear cells (PBMCs) stimulated by v/v% formalin-immobilized FA-immobilized influenza virions or deactivated influenza virions. Specifically, in the European Pharmacopoeia MONOCYTE-ACTIVATION TEST, human PBMC was used as a mixed pool with a minimum of 4 donors, and the measurement was performed by changing it to 1 donor. Table 2 shows the results of FA-immobilized influenza virion and the production of influenza virion-inactivated cytokines. It was found that the production of IL-6 in FA-immobilized influenza virions was considerably lower than that in deactivated influenza virions. It was thus taught that FA-immobilized influenza virions are safer than deactivated influenza virions.
[表2] [Table 2]
[實施例2] 物性評估 1.藉由蔗糖密度梯度離心法實行的分析 以遵照上述實施例1的方法,以流行性感冒病毒粒子(A/CA株)的蛋白質的最終濃度成為2500μg/mL的方式,稀釋獲得的級分而得到懸浮液。之後,以最終濃度成為0.12v/v%的方式添加福馬林到上述懸浮液,於25℃使之反應1週。藉由利用PBS來透析反應液,獲得了FA固定化流行性感冒病毒粒子。將獲得的FA固定化流行性感冒病毒粒子藉由蔗糖密度梯度離心法進行了分析。將檢體疊置於15~60%的蔗糖密度梯度,於4℃、18000rpm(57500(×g))進行了離心分離16小時。離心後,以每1級分0.6mL進行劃分,測定了各級分的蔗糖濃度、HA價及蛋白質濃度。A/CA株(H1N1亞型株)的結果顯示於表3。在裂解流感抗原,蛋白質廣泛分布於蔗糖濃度25~50%,顯示出病毒粒子正在分解。相對於此,在FA固定化流行性感冒病毒粒子,顯示出是在高蔗糖濃度(44.3%)以單一峰(粒子性)被劃分。關於HA活性是10240倍。[Example 2] Physical property evaluation 1. Analysis by Sucrose Density Gradient Centrifugation Following the method of Example 1 above, the obtained fraction was diluted so that the final concentration of influenza virus particle (A/CA strain) protein was 2500 μg/mL to obtain a suspension. Thereafter, formalin was added to the above-mentioned suspension so that the final concentration became 0.12 v/v%, and it was made to react at 25 degreeC for 1 week. FA-immobilized influenza virus particles were obtained by dialyzing the reaction solution with PBS. The obtained FA-immobilized influenza virus particles were analyzed by sucrose density gradient centrifugation. The sample was superimposed on a 15-60% sucrose density gradient, and centrifuged at 4°C, 18,000 rpm (57,500 (×g)) for 16 hours. After centrifugation, the fraction was divided into 0.6 mL fractions, and the sucrose concentration, HA value, and protein concentration of each fraction were measured. The results of the A/CA strain (H1N1 subtype strain) are shown in Table 3. In cleaved influenza antigens, the protein was widely distributed at sucrose concentrations of 25-50%, showing that the virions were being broken down. In contrast, influenza virus particles immobilized in FA showed a single peak (particle-like) at a high sucrose concentration (44.3%). About HA activity is 10240 times.
[表3] [table 3]
2.藉由電子顯微鏡實行的解析 為了更詳細調查上述FA固定化流行性感冒病毒粒子(A/CA株)的形狀,實施了藉由電子顯微鏡的觀察。使用戊二醛將檢體於室溫固定20分鐘。之後,在觀察用的經離子塗覆的片狀網(sheet mesh)(日新EM公司製造)上,放上經固定的檢體,靜置約60秒,利用2%磷鎢酸水溶液進行了負染色。將染色後的檢體,使用穿透式電子顯微鏡(FEI Company公司製造的TecnaiG2:加速電壓120kV)進行了觀察、拍攝。2. Analysis by electron microscope In order to examine the shape of the above-mentioned FA-immobilized influenza virus particles (A/CA strain) in more detail, observation with an electron microscope was carried out. Specimens were fixed with glutaraldehyde at room temperature for 20 minutes. After that, the fixed specimen was placed on an ion-coated sheet mesh (manufactured by Nisshin EM Co., Ltd.) for observation, left to stand for about 60 seconds, and performed with 2% phosphotungstic acid aqueous solution. Negative staining. The stained specimen was observed and photographed using a transmission electron microscope (TecnaiG2 manufactured by FEI Company: accelerating voltage 120 kV).
將FA固定化流行性感冒病毒粒子經以電子顯微鏡拍攝的相片顯示於第1圖。FA固定化流行性感冒病毒粒子,與去活化流行性感冒病毒粒子同樣地保有粒子結構。The electron microscope photographs of FA-immobilized influenza virus particles are shown in Fig. 1 . FA immobilizes influenza virions and maintains the particle structure similarly to deactivated influenza virions.
3.動態光散射 遵照實施例1的方法製作將A型流行性感冒病毒H3N2亞型株(A/NewYork/39/2012(X-233A)株,以下有時記載為「A/NY株」)及B型流行性感冒病毒維多利亞系統株(B/Brisbane/60/2008株,以下有時記載為「B/BR株」)成為起源之FA固定化流行性感冒病毒粒子,使用Zetasizer Nano ZS(Malvern公司製造)進行解析各自的平均粒徑。藉由動態光散射法測得的液體中的平均粒徑顯示於表4。FA固定化流行性感冒病毒粒子,其平均粒徑在140~150nm左右且為單峰性。由此可知,FA固定化流行性感冒病毒粒子的平均粒徑,與去活化流行性感冒病毒粒子同等。又,FA固定化流行性感冒病毒粒子保有粒子結構,沒有確認到如凝集體般的雜質。3. Dynamic Light Scattering According to the method of Example 1, a type A influenza virus H3N2 subtype strain (A/NewYork/39/2012 (X-233A) strain, hereinafter sometimes referred to as "A/NY strain") and type B influenza virus was produced. FA-immobilized influenza virus particles originating from influenza virus Victoria strain (B/Brisbane/60/2008 strain, hereinafter sometimes referred to as "B/BR strain") were analyzed using Zetasizer Nano ZS (manufactured by Malvern Co., Ltd.) respective average particle sizes. The average particle size in the liquid measured by the dynamic light scattering method is shown in Table 4. FA-immobilized influenza virus particles have an average particle size of about 140-150 nm and are unimodal. From this, it can be seen that the average particle size of FA-immobilized influenza virus particles is equivalent to that of deactivated influenza virus particles. Also, the FA-immobilized influenza virus particles maintained the particle structure, and impurities such as aggregates were not confirmed.
[表4] [Table 4]
4.分子量分布測定(SEC) 關於B型流行性感冒病毒的山形系統株(B/Massachusetts/02/2012(BX-51B)株(以下有時記載為「B/MA株」)),以遵照上述實施例1的方法製作了FA固定化流行性感冒病毒粒子(B/MA株)。關於A/CA株(H1N1亞型株)、A/NY株(H3N2亞型株)、及B/MA株(B型株),將裂解流感抗原、及FA固定化流行性感冒病毒粒子的分子量分布測定,使用尺寸篩除層析(商品名:TSKgel G6000PWXL(TOSOH公司製造))進行了分子量測定(溶析液中使用PBS,流速以0.5ml/min進行)。其溶出樣式顯示於表5。FA固定化流行性感冒病毒粒子在溶出時間16~17分鐘附近確認到單峰性的峰。另一方面,源自同一株的裂解流感抗原在溶出時間19~30分鐘附近確認到三峰性的峰。4. Molecular Weight Distribution Determination (SEC) The Yamagata strain of influenza B virus (B/Massachusetts/02/2012 (BX-51B) strain (hereinafter sometimes referred to as "B/MA strain")) was produced by following the method of Example 1 above FA immobilized influenza virus particles (B/MA strain). For A/CA strain (H1N1 subtype strain), A/NY strain (H3N2 subtype strain), and B/MA strain (B type strain), the molecular weight of split influenza antigen and FA-immobilized influenza virus particles For distribution measurement, molecular weight was measured using size exclusion chromatography (trade name: TSKgel G6000PWXL (manufactured by TOSOH)) (PBS was used for the eluent, and the flow rate was 0.5 ml/min). Its dissolution profile is shown in Table 5. The FA-immobilized influenza virus particles had a unimodal peak around the elution time of 16 to 17 minutes. On the other hand, for the split influenza antigen derived from the same strain, trimodal peaks were observed around the elution time of 19 to 30 minutes.
[表5] [table 5]
5.藉由交聯度的分析 針對在實施例1製備成的FA固定化流行性感冒病毒粒子(A/CA株),使用以下的順序,分析藉由固定劑的交聯度。首先,於檢體中添加SDS-Buffer(十二烷基磺酸鈉緩衝液)(終濃度:Tris(三羥甲基氨基甲烷)0.76w/v%,SDS(十二烷基磺酸鈉)1w/v%,Glycerol(甘油)10v/v%,BromophenolBlue(溴酚藍;BPB)0.01w/v%)與2-巰基乙醇(終濃度:0.8v/v%),煮沸6分鐘後,使用PAGEL NPU-12.5L(ATTO公司製造,商品名)或PAGEL NPU-R12.5L(ATTO公司製造,商品名),進行了SDS-PAGE(十二烷基磺酸鈉-聚丙烯醯胺膠體電泳)。電泳後,進行CBB(考馬斯亮藍;Coomassie Brilliant Blue)染色,於LAS3000(富士FILM股份有限公司製造,商品名)讀取影像。若藉由固定劑的交聯增進時,構成病毒的蛋白質之一的M1蛋白質,會從原本的泳帶的位置(25~37kDa)朝向高分子側位移。因此,教示了於原本的位置所檢測出的M1蛋白質的泳帶(M1泳帶)會變得稀薄,將此作為交聯度的指標。具體而言,算出了相對於沒有進行固定化的流行性感冒病毒粒子的M1泳帶的密度測定値而言,利用各福馬林濃度處理過的FA固定化流行性感冒病毒粒子於原本的位置所檢測出的M1泳帶的密度測定値的相對值(%)(以下亦有將此相對值記載為「M1蛋白質殘存率」(%)的情況)。結果顯示於表6。利用福馬林濃度0.05%、0.08%、0.11%、0.14%固定而成的FA固定化流行性感冒病毒粒子(A/CA株)的M1蛋白質殘存率,分別是35.7%、23.8%、11.0%、5.4%,教示了隨著福馬林濃度的提高,交聯受到促進,M1蛋白質殘存率會降低。5. By analyzing the degree of cross-linking For the FA-immobilized influenza virus particles (A/CA strain) prepared in Example 1, the degree of crosslinking by the fixative was analyzed using the following procedure. First, add SDS-Buffer (sodium dodecylsulfonate buffer) to the specimen (final concentration: Tris (trishydroxymethylaminomethane) 0.76w/v%, SDS (sodium dodecylsulfonate) 1w/v%, Glycerol (glycerol) 10v/v%, BromophenolBlue (bromophenol blue; BPB) 0.01w/v%) and 2-mercaptoethanol (final concentration: 0.8v/v%), after boiling for 6 minutes, use PAGEL NPU-12.5L (manufactured by ATTO, brand name) or PAGEL NPU-R12.5L (manufactured by ATTO, brand name), carried out SDS-PAGE (sodium dodecylsulfonate-polyacrylamide gel electrophoresis) . After electrophoresis, CBB (Coomassie Brilliant Blue) staining was performed, and images were read with LAS3000 (manufactured by Fuji Film Co., Ltd., trade name). When the cross-linking by the fixative increases, the M1 protein, one of the proteins constituting the virus, shifts from the original band position (25 to 37 kDa) toward the polymer side. Therefore, it was shown that the band of the M1 protein detected at the original position (M1 band) became thinner, and this was used as an index of the degree of cross-linking. Specifically, the density measurement value of the M1 band of influenza virus particles that were not immobilized was calculated according to the density of the immobilized influenza virus particles at their original positions using FA treated with various formalin concentrations. The relative value (%) of the density measurement value of the detected M1 swimming band (this relative value may also be described as "M1 protein remaining rate" (%) below). The results are shown in Table 6. The remaining rates of M1 protein in FA-immobilized influenza virus particles (A/CA strain) fixed with formalin concentrations of 0.05%, 0.08%, 0.11%, and 0.14% were 35.7%, 23.8%, 11.0%, and 5.4%, teaching that as the concentration of formalin increases, the cross-linking is promoted, and the residual rate of M1 protein will decrease.
[表6] [Table 6]
6.免疫原性1(小鼠肌肉內接種) 使用小鼠來評估FA固定化流行性感冒病毒粒子(A/CA株)中的免疫原性。於ddY小鼠(雌性,8週齡)中,將裂解流感抗原、去活化流行性感冒病毒粒子、或FA固定化流行性感冒病毒粒子(A/CA株),以蛋白質量為0.8μg的接種量進行了肌肉內接種(每一群為13隻)。於免疫3週後對小鼠進行全採血,並使之安樂死。藉由離心分離獲得血清,依照「病原體檢驗手冊」(國立感染症研究所編),測定了HI抗體力價。於小鼠進行了肌肉內接種時的免疫原性(HI抗體力價(GMT))的結果顯示於表7。FA固定化流行性感冒病毒粒子(A/CA株),與裂解流感抗原相比免疫原性較高,且是與去活化流行性感冒病毒粒子同等的免疫原性。6. Immunogenicity 1 (intramuscular inoculation in mice) Mice were used to assess immunogenicity in FA-immobilized influenza virions (A/CA strain). In ddY mice (female, 8 weeks old), split influenza antigen, deactivated influenza virions, or FA-immobilized influenza virions (A/CA strain) were inoculated with 0.8 μg of protein A small amount was inoculated intramuscularly (13 animals per group). Three weeks after immunization, the mice were bled and euthanized. Serum was obtained by centrifugation, and the HI antibody titer was determined according to the "Pathogen Test Manual" (edited by the National Institute of Infectious Diseases). Table 7 shows the results of immunogenicity (HI antibody titer (GMT)) when mice were intramuscularly inoculated. FA-immobilized influenza virions (A/CA strain) are more immunogenic than split influenza antigens, and are equally immunogenic as deactivated influenza virions.
[表7] [Table 7]
7.免疫原性2(小鼠皮內接種) 使用與上述「6.免疫原性1(小鼠肌肉內接種)」相同的順序,藉由小鼠皮內接種來評估FA固定化流行性感冒病毒粒子(A/CA株)中的免疫原性。於ddY小鼠(雌性,8週齡)中,將裂解流感抗原、去活化流行性感冒病毒粒子、或FA固定化流行性感冒病毒粒子,以0.2μg作為蛋白質量的接種量進行了皮內接種(每一群5隻)。於免疫3週後對小鼠進行全採血,並使之安樂死。藉由離心分離獲得血清,測定了HI抗體力價。於小鼠進行了皮內接種時的免疫原性(HI抗體力價(GMT))的結果顯示於表8。FA固定化流行性感冒病毒粒子,與裂解流感抗原相比為具顯著性差異地免疫原性較高,且與去活化流行性感冒病毒粒子同等的免疫原性。7. Immunogenicity 2 (intradermal inoculation in mice) Immunogenicity in FA-immobilized influenza virus particles (A/CA strain) was evaluated by intradermal inoculation of mice using the same procedure as in "6. Immunogenicity 1 (intramuscular inoculation of mice)" above . In ddY mice (female, 8 weeks old), split influenza antigen, deactivated influenza virions, or FA-immobilized influenza virions were inoculated intradermally at an inoculum volume of 0.2 μg as the protein amount (5 in each group). Three weeks after immunization, the mice were bled and euthanized. Serum was obtained by centrifugation, and the titer of HI antibody was determined. Table 8 shows the results of immunogenicity (HI antibody titer (GMT)) when mice were intradermally inoculated. FA-immobilized influenza virions were significantly more immunogenic than split influenza antigens, and were equally immunogenic as deactivated influenza virions.
[表8] [Table 8]
8.免疫原性3(食蟹獼猴皮下接種) 針對A/CA株(H1N1亞型株)、A/NY株(H3N2亞型株)、B/BR株及B/MA株(B型株),使用食蟹獼猴(雄性或雌性,17~25個月齡)來評估裂解流感抗原、去活化流行性感冒病毒粒子、及FA固定化流行性感冒病毒粒子的免疫原性。將裂解流感抗原、去活化流行性感冒病毒粒子、或FA固定化流行性感冒病毒粒子,於每1群為8或9隻以相當於7.5μgHA(相當於裂解流感抗原的HA量的蛋白質量)的接種量,在21日的間隔進行了兩次的皮下接種。於第2次免疫前、及第2次免疫後的第21日進行了部分採血。藉由離心分離獲得血清,依照「病原體檢驗手冊」(國立感染症研究所編),測定了HI抗體力價及中和抗體力價。將免疫原性的結果,顯示於表9(第2次免疫後第21日的HI抗體力價(GMT))、表10(第2次免疫後第21日的中和抗體力價(GMT))。在HI抗體力價,可知FA固定化流行性感冒病毒粒子與裂解流感抗原相比具有較高的免疫原性。尤其是,在B/MA株以外,與裂解流感抗原相比為具顯著性差異地免疫原性較高。即使在中和抗體力價中,與裂解流感抗原相比,FA固定化流行性感冒病毒粒子在全部的A型株及B型株中,皆為具顯著性差異地免疫原性較高。8. Immunogenicity 3 (subcutaneous inoculation in cynomolgus monkeys) For A/CA strain (H1N1 subtype strain), A/NY strain (H3N2 subtype strain), B/BR strain and B/MA strain (B type strain), use cynomolgus monkeys (male or female, 17-25 months) to evaluate the immunogenicity of split influenza antigens, deactivated influenza virions, and FA-immobilized influenza virions. Split influenza antigens, deactivated influenza virions, or FA-immobilized influenza virions, in each group of 8 or 9 animals with an amount equivalent to 7.5 μg HA (the amount of protein equivalent to the amount of HA for split influenza antigens) The amount of inoculum was subcutaneously inoculated twice at intervals of 21 days. Partial blood collection was performed before the second immunization and on the 21st day after the second immunization. Serum was obtained by centrifugation, and the titers of HI antibodies and neutralizing antibodies were determined according to the "Pathogen Test Manual" (edited by the National Institute of Infectious Diseases). The results of immunogenicity are shown in Table 9 (HI antibody titer (GMT) on the 21st day after the second immunization), Table 10 (neutralizing antibody titer (GMT) on the 21st day after the second immunization ). In terms of HI antibody potency, it can be seen that FA-immobilized influenza virus particles have higher immunogenicity than split influenza antigens. In particular, except for the B/MA strain, the immunogenicity was significantly higher than that of the split influenza antigen. Even in neutralizing antibody titers, FA-immobilized influenza virions had significantly higher immunogenicity in all type A strains and type B strains than split influenza antigens.
[表9] [Table 9]
[表10] [Table 10]
9.抗體亞型解析 作為抗體亞型解析,針對在上述「6.免疫原性1(小鼠肌肉內接種)」所獲得的A/CA株(H1N1亞型株)、及B/MA株(B型株),藉由酵素免疫測定(ELISA)法來測定小鼠的血清中所包含的對病毒抗原具特異性的IgG1及IgG2a抗體力價。其結果,FA固定化流行性感冒病毒粒子,顯示了誘導出比具抗原特異性的IgG1更多的具抗原特異性的IgG2a(表11、表12)。這與去活化流行性感冒病毒粒子是同樣的結果,即使是與裂解流感抗原的比較中,亦可見到較強的IgG2a誘導傾向。由此結果來看,FA固定化流行性感冒病毒粒子,與會活化體液性免疫但幾乎無法活化細胞性免疫的裂解流感抗原相比,能夠期待疫苗有效性的更加提升。9. Antibody subtype analysis For antibody subtype analysis, the A/CA strain (H1N1 subtype strain) and the B/MA strain (B type strain) obtained in the above "6. Immunogenicity 1 (intramuscular inoculation of mice)" were obtained by using The titers of IgG1 and IgG2a antibodies specific to viral antigens contained in mouse serum were determined by enzyme immunoassay (ELISA). As a result, FA-immobilized influenza virions showed that more antigen-specific IgG2a was induced than antigen-specific IgG1 (Table 11, Table 12). This is the same result as the inactivated influenza virion, and even in comparison with the split influenza antigen, a strong IgG2a induction tendency was observed. From these results, FA-immobilized influenza virions can be expected to be more effective in vaccines than split influenza antigens, which activate humoral immunity but hardly activate cellular immunity.
[表11] 將經裂解流感抗原免疫的小鼠血清,分別稀釋25600倍時的值當作1EU/mL。[Table 11] The serum of mice immunized with the split influenza antigen was diluted 25600 times as 1 EU/mL.
[表12] 將經裂解流感抗原免疫的小鼠血清,分別稀釋25600倍時的值當作1EU/mL。[Table 12] The serum of mice immunized with the split influenza antigen was diluted 25600 times as 1 EU/mL.
10.RNA釋放量的評估 針對以遵照實施例1的方法所製備成的FA固定化流行性感冒病毒粒子(A/NY株),用以下的順序來評估蛋白分解酶處理中的經時性的RNA釋放量。首先,利用PBS來稀釋FA固定化流行性感冒病毒粒子,加入SDS與Proteinase K(蛋白酶K)於55℃使之反應,經時性地萃取出RNA。在萃取RNA中,使用了TRIzol LS Reagent與PureLink RNA Mini Kit、PureLink DNase(invitrogen公司製造,商品名)。萃取出的RNA的含量,是利用Quant-iT RiboGreen RNA Reagent and Kit(invitrogen公司製造,商品名)來測定。各FA濃度的經時性的RNA含量顯示於表13。其結果,顯示出FA濃度依存性的RNA釋放被緩釋化。結果教示了藉由FA固定之RNA釋放的緩釋化,使發炎性細胞激素的產生被緩釋化,而孕育出高安全性。10. Evaluation of RNA release For the FA-immobilized influenza virus particles (A/NY strain) prepared in accordance with the method of Example 1, the time-dependent RNA release amount during proteolytic enzyme treatment was evaluated in the following procedure. First, FA-immobilized influenza virus particles were diluted with PBS, SDS and Proteinase K were added to react at 55°C, and RNA was extracted over time. For RNA extraction, TRIzol LS Reagent, PureLink RNA Mini Kit, and PureLink DNase (manufactured by Invitrogen, trade name) were used. The content of the extracted RNA was measured using Quant-iT RiboGreen RNA Reagent and Kit (manufactured by Invitrogen, trade name). Table 13 shows the RNA content over time for each FA concentration. As a result, the release of RNA showing FA concentration dependence was sustained. The results suggest that the release of FA-immobilized RNA is slowed to release the production of inflammatory cytokines, resulting in high safety.
[表13] [Table 13]
[實施例3] (1)GA固定化流行性感冒病毒粒子的製備 1.戊二醛(GA)處理 將A型流行性感冒病毒(H3N2亞型株(A/NY株))及B型流行性感冒病毒(B型維多利亞系統株(B/BR株))用與實施例1同樣的方法進行了培養、精製。以精製後的各流行性感冒病毒粒子的蛋白質的最終濃度成為1000μg/mL的方式,稀釋各流行性感冒病毒粒子,獲得懸浮液。其次,使用1w/v%GA溶液,以GA濃度成為0.016w/v%或成為0.008w/v%的方式稀釋。將上述懸浮液,與經稀釋的GA溶液(0.016w/v%或0.008w/v%)等量混合,於4℃使之反應3日。反應結束後,利用PBS來透析反應液,藉由除去GA,獲得固定化流行性感冒病毒粒子(以下有時記載為「GA固定化流行性感冒病毒粒子」)。藉由利用3隻兔子中的放熱反應之和所評估的放熱試驗、及刺激了人類PBMC時的發炎性細胞激素的產量的定量,評估了獲得的GA固定化流行性感冒病毒粒子(A/NY株及B/BR株)的放熱活性。[Example 3] (1) Preparation of GA-immobilized influenza virus particles 1. Glutaraldehyde (GA) Treatment Type A influenza virus (H3N2 subtype strain (A/NY strain)) and type B influenza virus (type B Victoria strain (B/BR strain)) were cultured in the same manner as in Example 1 ,refined. Each influenza virion was diluted so that the final protein concentration of each purified influenza virion was 1000 μg/mL to obtain a suspension. Next, using a 1w/v% GA solution, it was diluted so that the GA concentration would be 0.016w/v% or 0.008w/v%. The above suspension was mixed with the diluted GA solution (0.016w/v% or 0.008w/v%) in equal amounts, and reacted at 4°C for 3 days. After the reaction, the reaction solution was dialyzed against PBS to remove GA to obtain immobilized influenza virus particles (hereinafter sometimes referred to as "GA-immobilized influenza virus particles"). The obtained GA-immobilized influenza virions (A/NY strain and B/BR strain) exothermic activity.
2.放熱試驗 藉由與實施例1同樣的方法,進行了放熱試驗。用生理食鹽水稀釋去活化流行性感冒病毒粒子及GA固定化流行性感冒病毒粒子(A/NY株及B/BR株),將1mL中的蛋白質含量設在240μg作為試料。將上述試料,以每1kg體重為1mL的量接種到兔子上,觀察發熱的上昇,直到6小時後為止。A/NY株的3隻兔子的放熱反應之和(℃)顯示於表14,B/BR株的3隻兔子的放熱反應之和(℃)顯示於表15。2. Exothermic test By the same method as in Example 1, an exothermic test was performed. Deactivated influenza virions and GA-immobilized influenza virions (A/NY strain and B/BR strain) were diluted with normal saline, and the protein content in 1 mL was set at 240 μg as a sample. The above-mentioned sample was inoculated into rabbits in an amount of 1 mL per 1 kg of body weight, and the rise of fever was observed until 6 hours later. The sum (°C) of the exothermic responses of the 3 rabbits of the A/NY strain is shown in Table 14, and the sum (°C) of the exothermic responses of the 3 rabbits of the B/BR strain is shown in Table 15.
GA固定化流行性感冒病毒粒子,都沒有確認到1.3℃以上的放熱反應之和,以去活化流行性感冒病毒粒子為基準計,可見到降低了2.5℃以上或3℃以上的放熱反應之和。可知GA固定化流行性感冒病毒粒子,放熱反應之和相當低。由此亦教示了,與去活化流行性感冒病毒粒子相比,GA固定化流行性感冒病毒粒子具有較高安全性。GA-immobilized influenza virus particles did not confirm the sum of exothermic reactions above 1.3°C, but based on deactivated influenza virus particles, the sum of exothermic reactions was reduced by 2.5°C or above or 3°C . It can be seen that GA immobilizes influenza virus particles, and the sum of exothermic reactions is quite low. This also teaches that GA-immobilized influenza virions are safer than deactivated influenza virions.
[表14] [Table 14]
[表15] [Table 15]
3.發炎性細胞激素的產量的定量 以遵照歐洲藥典MONOCYTE-ACTIVATION TEST的方法,定量了利用GA固定化流行性感冒病毒粒子或去活化流行性感冒病毒粒子刺激了人類PBMC時的細胞激素(IL-1β)的產量。具體而言,歐洲藥典MONOCYTE-ACTIVATION TEST中的將人類PBMC以最少4位供給者份作為混合池來使用的部份,變更成1位供給者份,而進行了測定。GA固定化流行性感冒病毒粒子(A/NY株及B/BR株)的細胞激素的產量的結果顯示於表16、表17。可知與去活化流行性感冒病毒粒子相比,GA固定化流行性感冒病毒粒子的發炎性細胞激素的產量相當低。由此教示了,GA固定化流行性感冒病毒粒子,與裂解流感抗原同樣地具有高安全性。3. Quantification of Inflammatory Cytokines Production The production of cytokine (IL-1β) was quantified when human PBMCs were stimulated with GA-immobilized influenza virions or deactivated influenza virions by following the method of the European Pharmacopoeia MONOCYTE-ACTIVATION TEST. Specifically, in the European Pharmacopoeia MONOCYTE-ACTIVATION TEST, human PBMC was used as a mixed pool with a minimum of 4 donors, and the measurement was performed by changing it to 1 donor. Table 16 and Table 17 show the results of the production of cytokines in GA-immobilized influenza virus particles (A/NY strain and B/BR strain). It was found that the production of inflammatory cytokines in GA-immobilized influenza virions was considerably lower than that of deactivated influenza virions. From this, it was taught that GA-immobilized influenza virions are as highly safe as splitting influenza antigens.
[表16] [Table 16]
[表17] [Table 17]
[實施例4] 物性評估 用以下的方法評估了在上述實施例3所獲得的GA固定化流行性感冒病毒粒子的物性。[Example 4] Physical property evaluation The physical properties of the GA-immobilized influenza virus particles obtained in Example 3 above were evaluated by the following methods.
1.藉由電子顯微鏡實行的解析 為了進一步詳細調查GA固定化流行性感冒病毒粒子(A/NY株)的形狀,用與實施例2同樣的方法,實施了藉由電子顯微鏡的觀察。作為代表,是將以0.008w/v%的GA濃度於4℃使之反應3日後的GA固定化流行性感冒病毒粒子(A/NY株)所拍攝成的相片顯示於第2圖。GA固定化流行性感冒病毒粒子保有粒子結構,並沒有確認到粒子彼此結合而成的凝集體。1. Analysis by electron microscope In order to further investigate the shape of GA-immobilized influenza virus particles (A/NY strain) in detail, observation with an electron microscope was carried out in the same manner as in Example 2. As a representative, a photograph taken of GA-immobilized influenza virus particles (A/NY strain) after reacting at 4°C for 3 days at a GA concentration of 0.008w/v% is shown in Fig. 2 . The GA-immobilized influenza virus particles retained the particle structure, and aggregates in which the particles bonded to each other were not confirmed.
2.動態光散射 使用Zetasizer Nano ZS(Malvern公司製造)來解析GA固定化流行性感冒病毒粒子的平均粒徑。藉由動態光散射法測得的液體中的平均粒徑顯示於表18。GA固定化流行性感冒病毒粒子,是平均粒徑在130~160nm左右且為單峰性。由此確認到,GA固定化流行性感冒病毒粒子的平均粒徑,與成為起源之病毒粒子同等。亦即,可知GA固定化流行性感冒病毒粒子的平均粒徑為單一種沒有改變。又,GA固定化流行性感冒病毒粒子的粒子結構保持著,沒有確認到如凝集體般的雜質。2. Dynamic Light Scattering The average particle diameter of GA-immobilized influenza virus particles was analyzed using Zetasizer Nano ZS (manufactured by Malvern). The average particle size in the liquid measured by the dynamic light scattering method is shown in Table 18. GA-immobilized influenza virus particles have an average particle size of about 130-160 nm and are unimodal. From this, it was confirmed that the average particle size of GA-immobilized influenza virus particles was equivalent to that of the originating virus particles. In other words, it can be seen that the average particle diameter of GA-immobilized influenza virus particles does not change for a single species. Also, the particle structure of GA-immobilized influenza virus particles was maintained, and impurities such as aggregates were not confirmed.
[表18] [Table 18]
3.分子量分布測定(SEC) 用與實施例2同樣的方法(SEC)測定了GA固定化流行性感冒病毒粒子的分子量分布。其溶出樣式顯示於表19。裂解流感抗原是在溶出時間16~30分鐘附近辨認到四峰性的峰。另一方面,GA固定化流行性感冒病毒粒子是在溶出時間16~17分鐘附近辨認到單峰性的峰。3. Molecular Weight Distribution Determination (SEC) The molecular weight distribution of GA-immobilized influenza virus particles was measured by the same method (SEC) as in Example 2. Its dissolution profile is shown in Table 19. In the split influenza antigen, four peaks were recognized around the elution time of 16 to 30 minutes. On the other hand, for GA-immobilized influenza virus particles, a unimodal peak was recognized around the elution time of 16 to 17 minutes.
[表19] [Table 19]
4.藉由交聯度的分析 用與實施例2同樣的方法分析了GA固定化流行性感冒病毒粒子之利用固定劑的交聯度。B/BR株(B型維多利亞系統株)的結果顯示於表20。使用戊二醛濃度0.004w/v%、0.008w/v%固定而成的GA固定化流行性感冒病毒粒子的M1蛋白質殘存率,分別是23.8%、10.8%,可知隨著戊二醛濃度的提高,交聯受到促進,M1蛋白質殘存率會降低。4. By analyzing the degree of cross-linking In the same manner as in Example 2, the degree of cross-linking of GA-immobilized influenza virus particles using a fixative was analyzed. The results for the B/BR strain (type B Victoria strain) are shown in Table 20. The remaining rates of M1 protein in GA-immobilized influenza virions fixed with glutaraldehyde concentrations of 0.004w/v% and 0.008w/v% were 23.8% and 10.8%, respectively. Increase, the cross-linking is promoted, and the residual rate of M1 protein will be reduced.
[表20] [Table 20]
5.免疫原性(小鼠肌肉內接種) 使用以下的順序來評估GA固定化流行性感冒病毒粒子(B/BR株)中的免疫原性。首先,於ddY小鼠(雌性,8週齡)中,將裂解流感抗原、或GA固定化流行性感冒病毒粒子以蛋白質量為0.8μg的接種量進行了肌肉內接種(每一群為16隻)。於免疫3週後對小鼠進行全採血,並使之安樂死。藉由離心分離獲得血清,測定了中和抗體力價。作為代表,將B/BR株(B型維多利亞系統株)的免疫原性(中和抗體力價(GMT))的結果顯示於表21。B型株的情況,GA固定化流行性感冒病毒粒子,與裂解流感抗原相比免疫原性較高。5. Immunogenicity (intramuscular inoculation in mice) The following sequence was used to evaluate immunogenicity in GA immobilized influenza virions (B/BR strain). First, split influenza antigen or GA-immobilized influenza virions were inoculated intramuscularly with a protein amount of 0.8 μg in ddY mice (female, 8 weeks old) (16 mice per group) . Three weeks after immunization, the mice were bled and euthanized. Serum was obtained by centrifugation, and the neutralizing antibody titer was determined. Table 21 shows the results of immunogenicity (neutralizing antibody titer (GMT)) of the B/BR strain (type B Victoria strain) as a representative example. In the case of type B strains, GA-immobilized influenza virions were more immunogenic than split influenza antigens.
[表21] [Table 21]
6.抗體亞型解析 作為抗體亞型解析,是藉由ELISA法測定了在上述「5.免疫原性(小鼠肌肉內接種)」獲得的小鼠的血清中所包含的對流行性感冒病毒的抗原具特異性的IgG1及IgG2a抗體力價。其結果,與裂解流感抗原互相對照地,GA固定化流行性感冒病毒粒子顯示了誘導出比具抗原特異性的IgG1更多的具抗原特異性的IgG2a(表22)。這與去活化流行性感冒病毒粒子是同樣的結果。由此結果來看,GA固定化流行性感冒病毒粒子,與會活化體液性免疫但幾乎無法活化細胞性免疫的裂解流感抗原相比,能夠期待疫苗有效性的更加提升。 6. Antibody subtype analysis As an analysis of antibody subtypes, antibodies specific to influenza virus antigens contained in the serum of mice obtained in the above "5. Immunogenicity (intramuscular inoculation of mice)" were measured by ELISA. IgG1 and IgG2a antibody titers. As a result, GA-immobilized influenza virions showed the induction of more antigen-specific IgG2a than antigen-specific IgG1 compared to split influenza antigens (Table 22). This is the same result as deactivating influenza virions. From these results, GA-immobilized influenza virions can be expected to be more effective in vaccines than split influenza antigens, which activate humoral immunity but hardly activate cellular immunity.
〔實施例5〕 [Example 5]
將A型流行性感冒病毒(H3N2亞型株(A/NY株))及B型流行性感冒病毒(維多利亞系統株B/BR株)與實施例1同樣地進行了培養、精製。以精製後的各流行性感冒病毒粒子的蛋白質的最終濃度是A/NY株成為2500μg/mL、B/BR株成為500μg/mL的方式,稀釋獲得的級分,獲得懸浮液。將EDC溶液以成為0.1~4M的方式使用PBS進行階段稀釋,以最終濃度成為50~500mM的方式添加到懸浮液,於冰鎮(0℃),使之反應2~20小時。反應結束後,利用PBS來透析反應液,藉由除去EDC,獲得固定化流行性感冒病毒粒子(以下有時記載為「EDC固定化流行性感冒病毒粒子」)。藉由利用3隻兔子中的放熱反應之和所評估的放熱試驗、及刺激了人類PBMC時的發炎性細胞激素的產量的定量,評估了獲得的EDC固定化流行性感冒病毒粒子(A/NY株、B/BR株)的放熱活性。Influenza A virus (H3N2 subtype strain (A/NY strain)) and influenza B virus (Victoria strain B/BR strain) were cultured and purified in the same manner as in Example 1. The obtained fractions were diluted so that the final concentration of the purified influenza virion protein was 2500 μg/mL for the A/NY strain and 500 μg/mL for the B/BR strain to obtain a suspension. The EDC solution was diluted stepwise with PBS to 0.1 to 4M, added to the suspension so that the final concentration was 50 to 500 mM, and allowed to react for 2 to 20 hours under ice (0°C). After the reaction, the reaction solution was dialyzed with PBS to remove EDC to obtain immobilized influenza virus particles (hereinafter sometimes referred to as "EDC immobilized influenza virus particles"). The obtained EDC-immobilized influenza virions (A/NY strain, B/BR strain) exothermic activity.
2.放熱試驗 藉由與實施例1同樣的方法,進行了放熱試驗。用生理食鹽水稀釋EDC固定化流行性感冒病毒粒子,將1mL中的蛋白質含量設在240μg作為試料。EDC固定化流行性感冒病毒粒子,是使用了以50mM或500mM的EDC濃度,於冰鎮使之反應2小時後,利用PBS將反應液經過透析處理後的檢體(關於A/NY株亦實施了5mM的EDC濃度)。又,關於A/NY株,亦使用了以5mM的EDC濃度,於4℃使之反應20小時後,利用PBS將反應液經過透析處理後的檢體。將上述試料,以每1kg體重為1mL的量接種到兔子上,觀察發熱的上昇,直到6小時後為止。3隻兔子的放熱反應之和(℃)顯示於表23、表24。2. Exothermic test By the same method as in Example 1, an exothermic test was performed. EDC-immobilized influenza virus particles were diluted with physiological saline, and the protein content in 1 mL was set at 240 μg as a sample. EDC-immobilized influenza virus particles were prepared by using EDC at a concentration of 50mM or 500mM, reacting it on ice for 2 hours, and dialyzing the reaction solution with PBS. 5 mM EDC concentration). Also, for the A/NY strain, a sample obtained by reacting at 4° C. for 20 hours at an EDC concentration of 5 mM, and dialyzing the reaction solution with PBS was also used. The above-mentioned sample was inoculated into rabbits in an amount of 1 mL per 1 kg of body weight, and the rise of fever was observed until 6 hours later. The sum (° C.) of the exothermic responses of the three rabbits is shown in Table 23 and Table 24.
EDC固定化流行性感冒病毒粒子,在全部的EDC處理條件下,並沒有確認到1.3℃以上的放熱反應之和。由此亦教示了,EDC固定化流行性感冒病毒粒子,與裂解流感抗原同樣地具有高安全性。In EDC-immobilized influenza virus particles, the sum of exothermic reactions above 1.3°C was not confirmed under all the EDC treatment conditions. This also teaches that EDC immobilization of influenza virions is as safe as splitting influenza antigens.
[表23] [Table 23]
[表24] [Table 24]
3.定量發炎性細胞激素的產量 以遵照歐洲藥典MONOCYTE-ACTIVATION TEST的方法,定量了利用EDC固定化流行性感冒病毒粒子或去活化流行性感冒病毒粒子刺激人類PBMC時的細胞激素(IL-1β及IL-6)的產量。具體而言,歐洲藥典MONOCYTE-ACTIVATION TEST中的將人類PBMC以最少4位供給者份作為混合池來使用的部份,變更成1位供給者份,而進行了測定。A/NY株及B/BR株的EDC固定化流行性感冒病毒粒子及去活化流行性感冒病毒粒子的細胞激素的產量的結果顯示於表25、表26。可知EDC固定化流行性感冒病毒粒子,與去活化流行性感冒病毒粒子相比,發炎性細胞激素產量相當低。由此教示了,與去活化流行性感冒病毒粒子相比,EDC固定化流行性感冒病毒粒子較容易抑制副反應。3. Quantification of Inflammatory Cytokines Production In accordance with the European Pharmacopoeia MONOCYTE-ACTIVATION TEST method, the production of cytokines (IL-1β and IL-6) when stimulating human PBMC with EDC-immobilized influenza virions or deactivated influenza virions was quantified. Specifically, in the European Pharmacopoeia MONOCYTE-ACTIVATION TEST, human PBMC was used as a mixed pool with a minimum of 4 donors, and the measurement was performed by changing it to 1 donor. Table 25 and Table 26 show the results of the production of EDC-immobilized influenza virions of the A/NY strain and the B/BR strain and the production of cytokines that inactivate the influenza virions. It can be seen that EDC-immobilized influenza virions have considerably lower production of inflammatory cytokines than deactivated influenza virions. This teaches that EDC-immobilized influenza virions are more likely to suppress side effects than deactivated influenza virions.
[表25] [Table 25]
[表26] [Table 26]
[實施例6] 物性評估 用以下的方法評估了在上述實施例5所獲得的EDC固定化流行性感冒病毒粒子的物性。[Example 6] Physical property evaluation The physical properties of the EDC-immobilized influenza virus particles obtained in Example 5 above were evaluated by the following methods.
1.藉由蔗糖密度梯度離心法實行的分析 藉由與實施例2同樣的方法,將EDC固定化流行性感冒病毒粒子藉由蔗糖密度梯度離心法進行了分析。作為代表,將在50mM的EDC濃度,於冰鎮使之反應2小時後的EDC固定化流行性感冒病毒粒子(A/NY株(H3N2亞型株))的結果顯示於表27。在裂解流感抗原,蛋白質廣泛分布於蔗糖濃度25~50%,顯示出病毒粒子正在分解。相對於此,在EDC固定化流行性感冒病毒粒子,顯示出是在高蔗糖濃度(47.2%)以單一峰(粒子性)被劃分。關於HA活性是10240倍。1. Analysis by Sucrose Density Gradient Centrifugation By the same method as in Example 2, the EDC-immobilized influenza virus particles were analyzed by sucrose density gradient centrifugation. Table 27 shows the results of EDC-immobilized influenza virus particles (A/NY strain (H3N2 subtype strain)) at an EDC concentration of 50 mM reacted on ice for 2 hours as a representative example. In cleaved influenza antigens, the protein was widely distributed at sucrose concentrations of 25-50%, showing that the virions were being broken down. In contrast, the immobilized influenza virus particles in EDC showed a single peak (particle nature) at high sucrose concentration (47.2%). About HA activity is 10240 times.
[表27] [Table 27]
2.藉由電子顯微鏡實行的解析 為了詳細調查EDC固定化流行性感冒病毒粒子的形狀,用與實施例2同樣的方法,實施了藉由電子顯微鏡的觀察。作為代表,將在500mM的EDC濃度,於冰鎮使之反應2小時後的EDC固定化流行性感冒病毒粒子所拍攝的相片顯示於第3圖。EDC固定化流行性感冒病毒粒子保有粒子結構,並沒有確認到抗原彼此所結合而成的凝集體。2. Analysis by electron microscope In order to examine the shape of EDC-immobilized influenza virus particles in detail, observation with an electron microscope was carried out in the same manner as in Example 2. As a representative, photographs taken of EDC-immobilized influenza virus particles after reacting at an EDC concentration of 500 mM for 2 hours on ice are shown in FIG. 3 . The EDC-immobilized influenza virion retains the particle structure, and aggregates formed by the combination of antigens were not confirmed.
3.動態光散射 使用Zetasizer Nano ZS(Malvern公司製造)來解析EDC固定化流行性感冒病毒粒子的平均粒徑。藉由動態光散射法測得的液體中的平均粒徑顯示於表28。EDC固定化流行性感冒病毒粒子(A/NY株(H3N2亞型株)、B/BR株(B型株)),是平均粒徑在130~160nm左右且為單峰性。由此確認到,EDC固定化流行性感冒病毒粒子的平均粒徑,與成為起源之病毒粒子同等。亦即,可知即使經過EDC處理,固定化流行性感冒病毒粒子的平均粒徑仍為單一種沒有改變。又,EDC固定化流行性感冒病毒粒子的粒子結構保持著,沒有確認到如凝集體般的雜質。3. Dynamic Light Scattering The average particle size of EDC-immobilized influenza virus particles was analyzed using Zetasizer Nano ZS (manufactured by Malvern). The average particle size in the liquid measured by the dynamic light scattering method is shown in Table 28. EDC-immobilized influenza virus particles (A/NY strain (H3N2 subtype strain), B/BR strain (B type strain)) have an average particle size of about 130-160 nm and are unimodal. From this, it was confirmed that the average particle diameter of EDC-immobilized influenza virus particles was equivalent to that of the originating virus particles. That is, it can be seen that the average particle diameter of immobilized influenza virus particles remains unchanged for a single species even after EDC treatment. Also, the particle structure of EDC-immobilized influenza virus particles was maintained, and impurities such as aggregates were not confirmed.
[表28] [Table 28]
4.分子量分布測定(SEC) 測定了以H3N2亞型株(A/NY株)作為起源的EDC固定化流行性感冒病毒粒子的分子量分布。測定是使用尺寸篩除層析(商品名:TSKgel G6000PWXL(TOSOH公司製造))來進行(溶析液是使用PBS,流速是以0.5ml/min進行)。其溶出樣式顯示於表29。裂解流感抗原是在溶出時間16~30分鐘附近確認到四峰性的峰。另一方面,EDC固定化流行性感冒病毒粒子是在溶出時間16~17分鐘附近確認到單峰性的峰。4. Molecular Weight Distribution Determination (SEC) The molecular weight distribution of EDC-immobilized influenza virus particles originating from the H3N2 subtype strain (A/NY strain) was measured. The measurement was performed using size exclusion chromatography (trade name: TSKgel G6000PWXL (manufactured by TOSOH)) (PBS was used as the eluent, and the flow rate was 0.5 ml/min). Its dissolution profile is shown in Table 29. For the split influenza antigen, four peaks were confirmed around the elution time of 16 to 30 minutes. On the other hand, in the case of EDC-immobilized influenza virus particles, a unimodal peak was confirmed around the elution time of 16 to 17 minutes.
[表29] [Table 29]
5.藉由交聯度的分析 用與實施例2同樣的方法分析了藉由實施例5製備而成的EDC固定化流行性感冒病毒粒子之利用固定劑的交聯度。A/NY株(H3N2亞型株)的結果顯示於表30。利用EDC濃度50mM、500mM固定而成的EDC固定化流行性感冒病毒粒子的M1蛋白質殘存率(%),分別是85.7%、53.4%,可知隨著EDC濃度的提高,交聯受到促進,M1蛋白質殘存率會降低。B/BR株(B型維多利亞系統株)的結果顯示於表31。利用EDC濃度5mM、50mM、500mM固定而成的EDC固定化流行性感冒病毒粒子的M1蛋白質殘存率(%),分別是85.1%、56.1%、27.2%。可知與A/NY株(H3N2亞型株)同樣地,隨著EDC濃度的提高,交聯受到促進,M1蛋白質殘存率會降低。5. By analyzing the degree of cross-linking The cross-linking degree of the EDC-immobilized influenza virus particles prepared in Example 5 using the fixative was analyzed in the same manner as in Example 2. The results for the A/NY strain (H3N2 subtype strain) are shown in Table 30. The remaining rates (%) of M1 protein of EDC-immobilized influenza virus particles fixed by EDC concentrations of 50mM and 500mM were 85.7% and 53.4%, respectively. The survival rate will decrease. The results for the B/BR strain (type B Victoria strain) are shown in Table 31. The remaining rates (%) of M1 protein of EDC-immobilized influenza virions fixed by EDC concentrations of 5mM, 50mM, and 500mM were 85.1%, 56.1%, and 27.2%, respectively. Similar to the A/NY strain (H3N2 subtype strain), it can be seen that as the concentration of EDC increases, cross-linking is promoted, and the remaining rate of M1 protein decreases.
[表30] [Table 30]
[表31] [Table 31]
6.免疫原性(小鼠肌肉內接種) 使用小鼠來評估EDC固定化流行性感冒病毒粒子中的免疫原性。藉由與實施例4同樣的方法獲得血清,測定了HI抗體力價及中和抗體力價。作為代表,將以50mM及500mM的EDC濃度,於冰鎮使之反應2小時後的EDC固定化流行性感冒病毒粒子的免疫原性的結果,顯示於表32(A/NY株的HI抗體力價(GMT))、表33(A/NY株的中和抗體力價(GMT))及表34(B/BR株的中和抗體力價(GMT))。A/NY株(H3N2亞型株)、及B/BR株(B型維多利亞系統株)的情況,與裂解流感抗原相比,EDC固定化流行性感冒病毒粒子為具顯著性差異的高免疫原性。6. Immunogenicity (intramuscular inoculation in mice) Mice were used to assess immunogenicity in EDC-immobilized influenza virions. Serum was obtained by the same method as in Example 4, and the HI antibody titer and neutralizing antibody titer were measured. As a representative, the results of immunogenicity of EDC-immobilized influenza virus particles after reacting with EDC concentrations of 50 mM and 500 mM in ice for 2 hours are shown in Table 32 (HI antibody potency of A/NY strain (GMT)), Table 33 (A/NY strain neutralizing antibody titer (GMT)) and Table 34 (B/BR strain neutralizing antibody titer (GMT)). In the case of A/NY strain (H3N2 subtype strain) and B/BR strain (type B Victoria system strain), EDC-immobilized influenza virions are significantly different highly immunogens compared with split influenza antigens sex.
[表32] [Table 32]
[表33] [Table 33]
[表34] [Table 34]
7.抗體亞型解析 作為抗體亞型解析,藉由ELISA法來測定在上述「6.免疫原性(小鼠肌肉內接種)」所獲得的小鼠的血清中所包含的對病毒抗原具特異性的IgG1及IgG2a抗體力價。其結果,與裂解流感抗原互相對照地,EDC固定化流行性感冒病毒粒子顯示了誘導出比具抗原特異性的IgG1更多的具抗原特異性的IgG2a(表35、表36)。由此結果來看,EDC固定化流行性感冒病毒粒子,與會活化體液性免疫但幾乎無法活化細胞性免疫的裂解流感抗原相比,能夠期待疫苗有效性的更加提升。7. Antibody subtype analysis For antibody subtype analysis, IgG1 and IgG2a antibodies specific to viral antigens contained in the serum of mice obtained in the above "6. Immunogenicity (intramuscular inoculation of mice)" were measured by ELISA method power price. As a result, EDC-immobilized influenza virions induced more antigen-specific IgG2a than antigen-specific IgG1 compared to split influenza antigens (Table 35, Table 36). From these results, EDC-immobilized influenza virions can be expected to be more effective in vaccines than split influenza antigens, which activate humoral immunity but hardly activate cellular immunity.
[表35] 將經裂解流感抗原(IgG1)、類病毒粒子(IgG2a)免疫的小鼠血清,分別稀釋25600倍時的值當作1EU/mL。[Table 35] The serum of mice immunized with split influenza antigen (IgG1) and virus-like particles (IgG2a) was diluted 25600 times as 1 EU/mL.
[表36] 將經裂解流感抗原(IgG1)、類病毒粒子(IgG2a)免疫的小鼠血清,分別稀釋25600倍時的值當作1EU/mL。[Table 36] The serum of mice immunized with split influenza antigen (IgG1) and virus-like particles (IgG2a) was diluted 25600 times as 1 EU/mL.
8.免疫原性(食蟹獼猴皮下接種) 使用食蟹獼猴並用以下的順序來評估EDC固定化流行性感冒病毒粒子中的免疫原性。首先,對食蟹獼猴(雄性或雌性,29~35個月齡),將裂解流感抗原、或EDC固定化流行性感冒病毒粒子以HA含量為15μg的接種量進行了皮下接種(每一群為8隻)。間隔3週進行2次皮下接種,於第2次免疫後第4週進行了採血。藉由與實施例2同樣的方法獲得血清,測定了HI抗體力價及中和抗體力價。作為代表,以5mM的EDC濃度,於4℃反應20小時後的EDC固定化流行性感冒病毒粒子的免疫原性的HI抗體力價(GMT)的結果顯示於表37、中和抗體力價(GMT)的結果顯示於表38。在A/CA株(H1N1亞型株)、及A/NY株(H3N2亞型株)、B/MA株(B型山形系統)、B/BR株(B型維多利亞系統株)中,與裂解流感抗原相比,EDC固定化流行性感冒病毒粒子為具顯著性差異性地免疫原性較高。8. Immunogenicity (subcutaneous inoculation in cynomolgus monkeys) Immunogenicity in EDC-immobilized influenza virions was evaluated using cynomolgus monkeys with the following sequence. First, cynomolgus monkeys (male or female, 29-35 months of age) were inoculated subcutaneously with split influenza antigen or EDC-immobilized influenza virions at an inoculation volume of 15 μg of HA (8 for each group). only). Two subcutaneous inoculations were performed at intervals of 3 weeks, and blood was collected 4 weeks after the second immunization. Serum was obtained by the same method as in Example 2, and the HI antibody titer and neutralizing antibody titer were measured. As a representative, the results of the immunogenic HI antibody titer (GMT) of EDC-immobilized influenza virus particles after reacting at 4°C for 20 hours at an EDC concentration of 5 mM are shown in Table 37. Neutralizing antibody titers ( GMT) are shown in Table 38. In A/CA strain (H1N1 subtype strain), A/NY strain (H3N2 subtype strain), B/MA strain (B-type Yamagata system), B/BR strain (B-type Victoria system strain), and lysis EDC-immobilized influenza virions were significantly differentially more immunogenic than influenza antigens.
[表37] [Table 37]
[表38] [Table 38]
9.嚴格試驗前後的穩定性(藉由交聯度的分析) 將嚴格試驗前後的交聯度的變化作為指標,來評估藉由實施例5製備成的EDC固定化流行性感冒病毒粒子的穩定性。首先,藉由與實施例2同樣的方法算出M1蛋白質殘存率(%),然後,將嚴格試驗前的M1蛋白質殘存率當作100%的情況下,算出於37℃進行1週的嚴格試驗後的M1蛋白質殘存率的比例。A/NY株(H3N2亞型株)的結果顯示於表39。與嚴格試驗前相比,去活化流行性感冒病毒粒子的嚴格試驗後的M1蛋白質殘存率上昇了24%。另一方面,與嚴格試驗前相比,利用EDC濃度50、500mM固定而成的EDC固定化流行性感冒病毒粒子的嚴格試驗後的M1蛋白質殘存率分別減少18%,教示出比起去活化流行性感冒病毒粒子,變化率較小,且更穩定。9. Stability before and after strict test (by analysis of cross-linking degree) The stability of the EDC-immobilized influenza virus particles prepared in Example 5 was evaluated by using the change of the degree of cross-linking before and after the rigorous test as an index. First, the M1 protein remaining rate (%) was calculated by the same method as in Example 2, and then, when the M1 protein remaining rate before the rigorous test was regarded as 100%, it was calculated at 37°C for one week after the rigorous test The ratio of the M1 protein residual rate. The results for the A/NY strain (H3N2 subtype strain) are shown in Table 39. After the rigorous test of deactivated influenza virions, the residual rate of M1 protein increased by 24% compared with that before the rigorous test. On the other hand, compared with before the rigorous test, the M1 protein residual rate of the EDC-immobilized influenza virus particles immobilized with EDC concentrations of 50 and 500 mM was reduced by 18% after the rigorous test. Influenza virions have a smaller rate of change and are more stable.
[表39] [Table 39]
10.在嚴格試驗下的穩定性(藉由單向輻射免疫擴散試驗實行的分析) 將嚴格試驗前後的HA含量的變化作為指標,來評估藉由實施例5製備成的EDC固定化流行性感冒病毒粒子的穩定性。首先,藉由單向輻射免疫擴散試驗(生物學性製劑基準(日本國,厚生勞動省告示第192號))算出HA含量,然後,將嚴格試驗前的HA含量當作100%的情況下,算出於37℃進行1週的嚴格試驗後的HA含量的變化率。A/CA株(H1N1亞型株)、及A/NY株(H3N2亞型株)、B/MA株(B型山形系統)、B/BR株(B型維多利亞系統株)的結果顯示於表40。結果教示出EDC固定化流行性感冒病毒粒子的嚴格試驗後的HA含量的變化率較小,且穩定。10. Stability under strict test (analysis by one-way radiation immunodiffusion test) The stability of the EDC-immobilized influenza virus particles prepared in Example 5 was evaluated by using the change of the HA content before and after the rigorous test as an index. First, the HA content is calculated by the one-way radiation immunodiffusion test (biological agent standard (Japan, Ministry of Health, Labor and Welfare Notification No. 192)), and then, when the HA content before the rigorous test is regarded as 100%, The rate of change in the HA content after the severe test at 37° C. for one week was calculated. The results of A/CA strain (H1N1 subtype strain), A/NY strain (H3N2 subtype strain), B/MA strain (B type Yamagata system), and B/BR strain (B type Victoria system strain) are shown in the table 40. The results showed that the rate of change in the HA content after the rigorous test of EDC-immobilized influenza virions was small and stable.
[表40] [Table 40]
11.於5℃保存11個月下的穩定性(藉由交聯度的分析) 將於5℃保存11個月的前後的交聯度的變化作為指標來評估藉由實施例5製備成的EDC固定化流行性感冒病毒粒子的穩定性。首先,藉由與實施例2同樣的方法算出M1蛋白質殘存率(%),然後,將於5℃保存11個月前的M1蛋白質殘存率當作100%的情況下,算出於5℃保存11個月下的M1蛋白質殘存率的比例。A/CA株(H1N1亞型株)、及A/NY株(H3N2亞型株)、B/MA株(B型山形系統)、B/BR株(B型維多利亞系統株)的結果顯示於表41。結果教示出EDC固定化流行性感冒病毒粒子在於5℃保存11個月下的M1蛋白質殘存率的變化率小,且穩定。11. Stability at 5°C for 11 months (by analysis of cross-linking degree) The stability of the EDC-immobilized influenza virus particles prepared in Example 5 was evaluated using the change of cross-linking degree before and after storage at 5° C. for 11 months as an index. First, the M1 protein remaining rate (%) was calculated by the same method as in Example 2, and then, when the M1 protein remaining rate before storage at 5°C for 11 months was regarded as 100%, the remaining rate at 5°C for 11 months was calculated. The ratio of M1 protein residual rate under months. The results of A/CA strain (H1N1 subtype strain), A/NY strain (H3N2 subtype strain), B/MA strain (B type Yamagata system), and B/BR strain (B type Victoria system strain) are shown in the table 41. The results showed that the EDC-immobilized influenza virus particles had a small and stable change rate of the M1 protein remaining rate when stored at 5° C. for 11 months.
[表41] [Table 41]
12.於5℃保存11個月下的穩定性(藉由單向輻射免疫擴散試驗實行的分析) 將於5℃保存11個月前後的HA含量的變化作為指標,來評估藉由實施例5製備成的EDC固定化流行性感冒病毒粒子的穩定性。首先,藉由單向輻射免疫擴散試驗(生物學性製劑基準(日本國,厚生勞動省告示第192號))算出HA含量,然後,將於5℃保存11個月前的HA含量當作100%的情況下,算出於5℃保存11個月下的HA含量的變化率。A/CA株(H1N1亞型株)、及A/NY株(H3N2亞型株)、B/MA株(B型山形系統)、B/BR株(B型維多利亞系統株)的結果顯示於表42。結果教示出EDC固定化流行性感冒病毒粒子於5℃保存11個月下的HA含量的變化率小,且穩定。12. Stability at 5°C for 11 months (analysis by one-way radiation immunodiffusion assay) The stability of the EDC-immobilized influenza virus particles prepared in Example 5 was evaluated by using the change of HA content before and after storage at 5° C. for 11 months as an index. First, the HA content was calculated by the one-way radiation immunodiffusion test (biological preparation standard (Japan, Ministry of Health, Labor and Welfare Notification No. 192)), and then the HA content before storage at 5°C for 11 months was regarded as 100 %, calculate the rate of change in HA content stored at 5°C for 11 months. The results of A/CA strain (H1N1 subtype strain), A/NY strain (H3N2 subtype strain), B/MA strain (B type Yamagata system), and B/BR strain (B type Victoria system strain) are shown in the table 42. The results showed that the rate of change in the HA content of EDC-immobilized influenza virions stored at 5° C. for 11 months was small and stable.
[表42] [Table 42]
13.於5℃保存9個月下的穩定性(小鼠免疫原性(肌肉內接種)) 使用小鼠免疫原性(肌肉內接種)來評估藉由實施例5製備成的EDC固定化流行性感冒病毒粒子於5℃保存9個月下的穩定性。藉由與實施例4同樣的方法獲得血清,並測定HI抗體力價及中和抗體力價。作為代表,以5mM的EDC濃度,並於4℃反應20小時後的EDC固定化流行性感冒病毒粒子的免疫原性的HI抗體力價(GMT)的結果顯示於表43,中和抗體力價(GMT)的結果顯示於表44。在A/CA株(H1N1亞型株)、及A/NY株(H3N2亞型株)、B/MA株(B型山形系統)、B/BR株(B型維多利亞系統株)中,與裂解流感抗原相比,EDC固定化流行性感冒病毒粒子的免疫原性較高。13. Stability at 5°C for 9 months (immunogenicity in mice (intramuscular inoculation)) Mouse immunogenicity (intramuscular inoculation) was used to evaluate the stability of the EDC-immobilized influenza virus particles prepared in Example 5 when stored at 5°C for 9 months. Serum was obtained by the same method as in Example 4, and the HI antibody titer and neutralizing antibody titer were determined. As a representative, the results of the immunogenic HI antibody titer (GMT) of EDC-immobilized influenza virus particles after reacting at 4°C for 20 hours at an EDC concentration of 5 mM are shown in Table 43, neutralizing antibody titer (GMT) results are shown in Table 44. In A/CA strain (H1N1 subtype strain), A/NY strain (H3N2 subtype strain), B/MA strain (B-type Yamagata system), B/BR strain (B-type Victoria system strain), and lysis Compared with influenza antigen, the immunogenicity of EDC-immobilized influenza virion is higher.
[表43] [Table 43]
[表44] [Table 44]
14.於5℃保存9個月下的穩定性(抗體亞型) 作為抗體亞型解析,藉由ELISA法來測定在上述「13.於5℃保存9個月下的穩定性(小鼠免疫原性(肌肉內接種))」所獲得的小鼠的血清中所包含的對病毒抗原具特異性的IgG1及IgG2a抗體力價。其結果,即使是於5℃保存9個月後,與裂解流感抗原互相對照地,EDC固定化流行性感冒病毒粒子顯示了誘導出比具抗原特異性的IgG1更多的具抗原特異性的IgG2a(表45)。由此結果來看,能夠期待EDC固定化流行性感冒病毒粒子所活化的細胞性免疫,於5℃保存9個月後亦維持著。14. Stability at 5°C for 9 months (antibody isotype) For analysis of antibody subtypes, ELISA method was used to measure the serum concentration of mice obtained in the above "13. Stability at 5°C for 9 months (immunogenicity in mice (intramuscular inoculation))". Contains IgG1 and IgG2a antibody titers specific to viral antigens. As a result, EDC-immobilized influenza virions were shown to induce more antigen-specific IgG2a than antigen-specific IgG1 even after 9 months of storage at 5°C in comparison with split influenza antigens. (Table 45). From these results, it can be expected that the cellular immunity activated by EDC-immobilized influenza virus particles is maintained even after storage at 5°C for 9 months.
[表45] [Table 45]
15.於5℃保存10個月下的穩定性(食蟹獼猴免疫原性(皮下接種)) 使用食蟹獼猴免疫原性(皮下接種)來評估藉由實施例5製備成的EDC固定化流行性感冒病毒粒子於5℃保存10個月下的穩定性。首先,對食蟹獼猴(雄性或雌性,29~35個月齡),將裂解流感抗原、EDC固定化流行性感冒病毒粒子、及去活化流行性感冒病毒粒子以HA含量為15μg的接種量進行了皮下接種(每一群為8隻)。間隔3週進行2次皮下接種,於第2次免疫後第4週進行了採血。藉由與實施例2同樣的方法獲得血清,測定了HI抗體力價及中和抗體力價。作為代表,以5mM的EDC濃度,於4℃反應20小時後的EDC固定化流行性感冒病毒粒子於5℃保存10個月下的HI抗體力價(GMT)的結果顯示於表46,中和抗體力價(GMT)的結果顯示於表47(表46、表47分別為表37、表38的再次揭示)。在A/CA株(H1N1亞型株)、及A/NY株(H3N2亞型株)、B/MA株(B型山形系統)、B/BR株(B型維多利亞系統株)中,與裂解流感抗原相比,EDC固定化流行性感冒病毒粒子為具顯著性差異性地免疫原性較高。15. Stability at 5°C for 10 months (cynomolgus monkey immunogenicity (subcutaneous inoculation)) Cynomolgus monkey immunogenicity (subcutaneous inoculation) was used to evaluate the stability of the EDC-immobilized influenza virus particles prepared in Example 5 stored at 5°C for 10 months. First, cynomolgus monkeys (male or female, aged 29 to 35 months) were inoculated with split influenza antigen, EDC-immobilized influenza virions, and deactivated influenza virions with an HA content of 15 μg. subcutaneous inoculation (8 animals per group). Two subcutaneous inoculations were performed at intervals of 3 weeks, and blood was collected 4 weeks after the second immunization. Serum was obtained by the same method as in Example 2, and the HI antibody titer and neutralizing antibody titer were measured. As a representative, the results of HI antibody titer (GMT) of EDC-immobilized influenza virus particles after reacting at 4°C for 20 hours at an EDC concentration of 5 mM and storing at 5°C for 10 months are shown in Table 46. The results of the antibody titer (GMT) are shown in Table 47 (Table 46 and Table 47 are re-disclosures of Table 37 and Table 38, respectively). In A/CA strain (H1N1 subtype strain), A/NY strain (H3N2 subtype strain), B/MA strain (B-type Yamagata system), B/BR strain (B-type Victoria system strain), and lysis EDC-immobilized influenza virions were significantly differentially more immunogenic than influenza antigens.
[表46] [Table 46]
[表47] [Table 47]
16.RNA釋放量的評估 評估了藉由實施例5製備成的EDC固定化流行性感冒病毒粒子的蛋白分解酶處理中的經時性RNA釋放量。首先,利用PBS來稀釋去活化流行性感冒病毒粒子及EDC固定化流行性感冒病毒粒子,加入SDS與Proteinase K於55℃使之反應,經時性地萃取出RNA。在萃取RNA中,使用了TRIzol LS Reagent與PureLink RNA Mini Kit、PureLink DNase(invitrogen公司製造,商品名)。萃取出的RNA的含量,是利用Quant-iT RiboGreen RNA Reagent and Kit(invitrogen公司製造,商品名)來測定。作為代表,A/NY株(H3N2亞型株)的結果顯示於表48。其結果,與去活化流行性感冒病毒粒子相比,顯示出EDC固定化流行性感冒病毒粒子的RNA釋放正被緩釋化。16. Evaluation of RNA release The amount of RNA released over time during proteolytic enzyme treatment of the EDC-immobilized influenza virions prepared in Example 5 was evaluated. First, PBS was used to dilute deactivated influenza virions and EDC-immobilized influenza virions, and SDS and Proteinase K were added to react at 55°C, and RNA was extracted over time. For RNA extraction, TRIzol LS Reagent, PureLink RNA Mini Kit, and PureLink DNase (manufactured by Invitrogen, trade name) were used. The content of the extracted RNA was measured using Quant-iT RiboGreen RNA Reagent and Kit (manufactured by Invitrogen, trade name). As a representative, the results of the A/NY strain (H3N2 subtype strain) are shown in Table 48. As a result, it was shown that the release of RNA from EDC-immobilized influenza virions was slowed down compared to deactivated influenza virions.
[表48] [Table 48]
17.發炎性細胞激素產量的評估 評估了藉由實施例5製備成的EDC固定化流行性感冒病毒粒子的發炎性細胞激素的經時性產量。方法是遵照歐洲藥典MONOCYTE-ACTIVATION TEST。具體而言,歐洲藥典MONOCYTE-ACTIVATION TEST中的將人類PBMC以最少4位供給者份作為混合池來使用的部份,變更成1位供給者進行了測定。A/NY株(H3N2亞型株)中的去活化流行性感冒病毒粒子及EDC固定化流行性感冒病毒粒子的IL-1β產量的經時性變化顯示於表49,IL-6產量的經時性變化顯示於表50。又,B/BR株(B型維多利亞系統株)中的去活化流行性感冒病毒粒子及EDC固定化流行性感冒病毒粒子的IL-1β產量的經時性變化顯示於表51,IL-6產量的經時性變化顯示於表52。其結果,與去活化流行性感冒病毒粒子相比,顯示出EDC固定化流行性感冒病毒粒子的發炎性細胞激素的產生正被緩釋化。換句話說,藉由EDC固定,在體內的RNA釋放被緩釋化,使得發炎性細胞激素的產生延遲,故教示了EDC固定化流行性感冒病毒粒子具有高安全性。17. Assessment of Inflammatory Cytokines Production The time-dependent production of inflammatory cytokines of the EDC-immobilized influenza virions prepared in Example 5 was evaluated. The method is to comply with the European Pharmacopoeia MONOCYTE-ACTIVATION TEST. Specifically, in the European Pharmacopoeia MONOCYTE-ACTIVATION TEST, human PBMCs were used as a mixed pool with at least 4 donors, and the measurement was performed by changing to 1 donor. The time-dependent changes in IL-1β production of deactivated influenza virions and EDC-immobilized influenza virions in the A/NY strain (H3N2 subtype strain) are shown in Table 49, and the time-dependent changes in IL-6 production Sexual changes are shown in Table 50. In addition, the time-dependent changes in IL-1β production of deactivated influenza virions and EDC-immobilized influenza virions in the B/BR strain (B-type Victoria strain) are shown in Table 51. IL-6 production The time-course changes of are shown in Table 52. As a result, it was shown that the production of inflammatory cytokines in EDC-immobilized influenza virions was slowed down compared to deactivated influenza virions. In other words, by immobilizing EDC, the release of RNA in the body is slowed down, so that the production of inflammatory cytokines is delayed, so it is taught that EDC-immobilized influenza virus particles have high safety.
[表49] [Table 49]
[表50] [Table 50]
[表51] [Table 51]
[表52] [Table 52]
[實施例7] GA固定化日本腦炎病毒粒子的製備 1.戊二醛處理(對病毒粒子進行固定化粒子結構的步驟) 對Vero細胞培養日本腦炎疫苗原料藥(化學及血清療法研究所製造,商品名「ENCEVAC」,包含蛋白質濃度為60~90μg/ml的經利用0.08v/v%福馬林去活化完成的日本腦炎病毒粒子。以下有時記載為「去活化日本腦炎病毒粒子」),以最終濃度成為0.005~0.02w/v%的方式添加戊二醛,於4℃使之反應3日。反應結束後,利用類PBS溶液(添加了活化劑也就是乳糖(終濃度5w/v%)的PBS)來透析獲得的反應液。藉由透析,去除戊二醛,藉此獲得了固定化日本腦炎病毒粒子(以下有時記載為「GA固定化日本腦炎病毒粒子」)。藉由利用3隻兔子中的放熱反應之和所評估的放熱試驗、刺激了人類PBMC時的發炎性細胞激素的產量的定量,評估了獲得的GA固定化日本腦炎病毒粒子的放熱活性。[Example 7] Preparation of GA immobilized Japanese encephalitis virus particles 1. Glutaraldehyde treatment (the step of immobilizing the particle structure of virus particles) Japanese encephalitis vaccine raw material (manufactured by the Institute of Chemistry and Serotherapy, trade name "ENCEVAC") was cultured on Vero cells, containing Japanese encephalitis deactivated with 0.08v/v% formalin at a protein concentration of 60-90μg/ml Inflammation virus particles. Hereinafter, it may be described as "deactivated Japanese encephalitis virus particles"), glutaraldehyde was added so that the final concentration became 0.005 to 0.02 w/v%, and reacted at 4°C for 3 days. After the reaction, the obtained reaction solution was dialyzed with a PBS-like solution (PBS added with an activating agent, ie, lactose (final concentration 5w/v%)). Glutaraldehyde was removed by dialysis to obtain immobilized Japanese encephalitis virions (hereinafter sometimes referred to as "GA-immobilized Japanese encephalitis virions"). The exothermic activity of the obtained GA-immobilized Japanese encephalitis virions was evaluated by quantification of the production of inflammatory cytokines when human PBMCs were stimulated using an exothermic test evaluated by the sum of exothermic responses in 3 rabbits.
2.放熱試驗 藉由與實施例1同樣的方法,進行了放熱試驗。用生理食鹽水稀釋GA固定化日本腦炎病毒粒子或去活化日本腦炎病毒粒子,將1mL中的蛋白質含量設在70μg作為試料。將上述試料,以每1kg體重為3mL的量接種到兔子,觀察發熱的上昇,直到6小時後為止。3隻兔子的放熱反應之和(℃)顯示於表53。作為代表,評估了以0.01w/v%的戊二醛濃度於4℃反應3日後的GA固定化日本腦炎病毒粒子。2. Exothermic test By the same method as in Example 1, an exothermic test was performed. GA-immobilized Japanese encephalitis virus particles or deactivated Japanese encephalitis virus particles were diluted with physiological saline, and the protein content in 1 mL was set at 70 μg as a sample. The above-mentioned sample was inoculated into rabbits in an amount of 3 mL per 1 kg of body weight, and the rise of fever was observed until 6 hours later. The sum (°C) of the exothermic responses of the 3 rabbits is shown in Table 53. As a representative example, GA-immobilized Japanese encephalitis virions after reacting at 4° C. for 3 days at a glutaraldehyde concentration of 0.01 w/v% were evaluated.
GA固定化日本腦炎病毒粒子,沒有確認到1.3℃以上的放熱反應之和,與去活化日本腦炎病毒粒子相比,可見到降低了1.6℃以上的放熱反應之和。由此亦教示了,GA固定化日本腦炎病毒粒子具有高安全性。GA immobilized Japanese encephalitis virions, and the sum of exothermic reactions of 1.3°C or higher was not confirmed, and the sum of exothermic reactions was reduced by 1.6°C or higher compared with deactivated Japanese encephalitis virions. This also teaches that GA-immobilized Japanese encephalitis virus particles have high safety.
[表53] [Table 53]
3.定量發炎性細胞激素的產量 以遵照歐洲藥典MONOCYTE-ACTIVATION TEST的方法,定量了利用GA固定化日本腦炎病毒粒子或去活化日本腦炎病毒粒子刺激了人類PBMC時的細胞激素(IL-1β及IL-6)的產量。具體而言,歐洲藥典MONOCYTE-ACTIVATION TEST中的將人類PBMC以最少4位供給者份作為混合池來使用的部份,變更成1位供給者份,而進行了測定。其結果顯示於表54。結果可知,與去活化日本腦炎病毒粒子相比,GA固定化日本腦炎病毒粒子的發炎性細胞激素的產量相當低。由此教示了,與去活化日本腦炎病毒粒子相比,GA固定化日本腦炎病毒粒子具有較高安全性。3. Quantification of Inflammatory Cytokines Production The production of cytokines (IL-1β and IL-6) was quantified when human PBMCs were stimulated with GA-immobilized Japanese encephalitis virions or deactivated Japanese encephalitis virions by following the method of the European Pharmacopoeia MONOCYTE-ACTIVATION TEST. Specifically, in the European Pharmacopoeia MONOCYTE-ACTIVATION TEST, human PBMC was used as a mixed pool with a minimum of 4 donors, and the measurement was performed by changing it to 1 donor. The results are shown in Table 54. As a result, it was found that the production of inflammatory cytokines in GA-immobilized Japanese encephalitis virions was considerably lower than that of deactivated Japanese encephalitis virions. It was thus taught that GA-immobilized Japanese encephalitis virions are safer than deactivated Japanese encephalitis virions.
[表54] [Table 54]
[實施例8] 物性評估 用以下的方法評估了在上述實施例7所獲得的GA固定化日本腦炎病毒粒子的物性。[Example 8] Physical property evaluation The physical properties of the GA-immobilized Japanese encephalitis virus particles obtained in Example 7 above were evaluated by the following methods.
1.藉由電子顯微鏡實行的解析 為了詳細調查GA固定化日本腦炎病毒粒子的形狀,用與實施例2同樣的方法,實施了藉由電子顯微鏡的觀察。作為代表,是將以0.01w/v%的戊二醛濃度於4℃反應3日後的GA固定化日本腦炎病毒粒子所拍攝成的相片顯示於第4圖。GA固定化日本腦炎病毒粒子,因為利用戊二醛固定,所以與去活化日本腦炎病毒粒子同樣地保有粒子結構。1. Analysis by electron microscope In order to investigate the shape of GA-immobilized Japanese encephalitis virus particles in detail, observation with an electron microscope was carried out in the same manner as in Example 2. As a representative, a photograph taken of GA-immobilized Japanese encephalitis virus particles after reacting at a concentration of 0.01w/v% glutaraldehyde at 4°C for 3 days is shown in Fig. 4 . GA-immobilized Japanese encephalitis virions retain their particle structure similarly to deactivated Japanese encephalitis virions because they are immobilized with glutaraldehyde.
2.動態光散射 使用Zetasizer Nano ZS(Malvern公司製造)來解析GA固定化日本腦炎病毒粒子的平均粒徑。藉由動態光散射法測得的液體中的平均粒徑顯示於表55。GA固定化日本腦炎病毒粒子,是平均粒徑為約90nm且為單峰性。另一方面,去活化日本腦炎病毒粒子,是約80nm且為單峰性。又,GA固定化日本腦炎病毒粒子的粒子結構保持著,沒有確認到如凝集體般的雜質。2. Dynamic Light Scattering The average particle diameter of GA-immobilized Japanese encephalitis virus particles was analyzed using Zetasizer Nano ZS (manufactured by Malvern). The average particle size in the liquid measured by the dynamic light scattering method is shown in Table 55. GA-immobilized Japanese encephalitis virus particles have an average particle diameter of about 90 nm and are unimodal. On the other hand, the inactivated Japanese encephalitis virions were about 80 nm and unimodal. Also, the particle structure of GA-immobilized Japanese encephalitis virus particles was maintained, and impurities such as aggregates were not confirmed.
[表55] [Table 55]
3.分子量分布測定(SEC)
測定了GA固定化日本腦炎病毒粒子的分子量分布。測定是使用尺寸篩除層析(商品名:Superose 6 10/300 GE(GE Healthcare公司製造))來進行(溶析液是使用PBS,流速是以0.5ml/min進行)。其溶出樣式顯示於表56。GA固定化日本腦炎病毒粒子是在溶出時間14~15分鐘附近確認到單峰性的主峰。又,去活化日本腦炎病毒粒子亦在溶出時間14~15分鐘附近確認到單峰性的主峰。3. Molecular Weight Distribution Determination (SEC)
The molecular weight distribution of GA-immobilized Japanese encephalitis virus particles was determined. The measurement was performed using size exclusion chromatography (trade name:
[表56] [Table 56]
4.抗原的含量 藉由使用了抗日本腦炎病毒抗體的三明治ELISA法(三明治酵素免疫測定法),使用以下的順序來測定抗原的含量(抗原含量)。檢體中所包含的E抗原,會被結合有抗日本腦炎病毒兔子IgG(一次抗體;多株抗體)的微量盤捕捉。之後,藉由使結合了源自辣根之過氧化酶(HRP)的抗日本腦炎病毒E蛋白質單株抗體(二次抗體;單株抗體)反應,形成結合在微量盤的抗E抗原抗體/E抗原/二次抗體的複合體。藉由洗淨來除去未反應而殘留的試劑及檢體,使之與酵素基質液(鄰苯二胺溶液:OPD溶液)反應後,E抗原複合體上的HRP產生反應,引起發色。利用OPD的發色強度是與複合體的量(反映E抗原量)成比例的現象,測定出E抗原含量(抗原含量)。4. Antigen content The antigen content (antigen content) was measured by the sandwich ELISA method (sandwich enzyme immunoassay method) using the anti-Japanese encephalitis virus antibody using the following procedure. The E antigen contained in the specimen is captured by a microtiter plate to which anti-Japanese encephalitis virus rabbit IgG (primary antibody; polyclonal antibody) is bound. Then, by reacting the anti-Japanese encephalitis virus E protein monoclonal antibody (secondary antibody; monoclonal antibody) conjugated with horseradish-derived peroxidase (HRP), the anti-E antigen antibody bound to the microplate was formed /E antigen/secondary antibody complex. The remaining unreacted reagents and samples are removed by washing, and after reacting with the enzyme substrate solution (o-phenylenediamine solution: OPD solution), the HRP on the E antigen complex reacts to cause color development. Using the phenomenon that the color intensity of OPD is proportional to the amount of the complex (reflecting the amount of E antigen), the E antigen content (antigen content) is measured.
GA固定化日本腦炎病毒粒子及去活化日本腦炎病毒粒子各自的抗原含量顯示於表57。作為代表,將以0.01w/v%的戊二醛濃度於4℃反應3日後的GA固定化日本腦炎病毒粒子進行評估後,其是與去活化日本腦炎病毒粒子包含了同等的抗原。Table 57 shows the respective antigen contents of GA-immobilized Japanese encephalitis virions and deactivated Japanese encephalitis virions. As a representative example, GA-immobilized Japanese encephalitis virions after reacting at 4°C for 3 days at a glutaraldehyde concentration of 0.01w/v% were evaluated to contain the same antigen as deactivated Japanese encephalitis virions.
[表57] [Table 57]
5.藉由比活性實行的分析 針對在實施例7製備成的GA固定化日本腦炎病毒粒子,藉由比活性(抗原含量/蛋白質含量),評估了利用固定劑的交聯度。具體而言,使用於抗原含量的測定的單株抗體(503),會辨識中和抗原決定位,中和抗原決定位的結構如果變化,比活性就會下降。算出相對於沒有進行固定化的去活化日本腦炎病毒粒子的比活性而言,經各戊二醛濃度處理後的GA固定化日本腦炎病毒粒子的比活性的相對值(%)(以下有時將此相對值記載為「503抗體反應率」(%))。結果顯示於表58。利用戊二醛濃度0.005w/v%、0.01w/v%、0.02w/v%固定而成的GA固定化日本腦炎病毒粒子的503抗體反應率,分別為95.1%、74.7%、55.2%,教示出隨著戊二醛濃度升高,交聯受到促進,因503抗體抗原決定位的結構變化導致反應率的下降。5. Analysis by Specific Activity For the GA-immobilized Japanese encephalitis virus particles prepared in Example 7, the degree of cross-linking using a fixative was evaluated by specific activity (antigen content/protein content). Specifically, the monoclonal antibody (503) used for the determination of the antigen content recognizes the neutralizing epitope, and if the structure of the neutralizing epitope changes, the specific activity will decrease. Calculate the relative value (%) of the specific activity of the GA-immobilized Japanese encephalitis virion treated with each concentration of glutaraldehyde with respect to the specific activity of the deactivated Japanese encephalitis virion that was not immobilized (the following is This relative value is recorded as "503 antibody reaction rate" (%)). The results are shown in Table 58. The 503 antibody reaction rates of GA-immobilized Japanese encephalitis virus particles fixed by glutaraldehyde concentrations of 0.005w/v%, 0.01w/v%, and 0.02w/v% were 95.1%, 74.7%, and 55.2%, respectively. , taught that as the concentration of glutaraldehyde increased, cross-linking was promoted, resulting in a decrease in reactivity due to structural changes in the epitope of the 503 antibody epitope.
[表58] [Table 58]
6.免疫原性(小鼠腹腔內接種) 作為代表,是對ddY小鼠(雌性,4週齡),將利用0.005或者0.01w/v%的戊二醛濃度於4℃反應3日後的GA固定化日本腦炎病毒粒子或去活化日本腦炎病毒粒子以1μg或0.25μg的接種量進行了腹腔內接種(每一群為10隻)。免疫1週間後再次進行免疫,在該1週後將小鼠進行全採血,並使之安樂死。藉由離心分離獲得血清,每群等量作為混合池,依照「病原體檢驗手冊」(國立感染症研究所編)測定了中和抗體力價。由50%溶斑減少率算出的結果顯示於表59。與去活化日本腦炎病毒粒子相比,GA固定化日本腦炎病毒粒子為同等或在其以上的中和抗體力價。6. Immunogenicity (intraperitoneal inoculation of mice) As a representative, for ddY mice (female, 4 weeks old), GA-immobilized Japanese encephalitis virions or deactivated Japanese encephalitis virions were reacted at a concentration of 0.005 or 0.01w/v% glutaraldehyde at 4°C for 3 days. Infection virus particles were inoculated intraperitoneally at an inoculation volume of 1 μg or 0.25 μg (10 animals per group). Immunization was performed again one week after the immunization, and the whole mouse was blood-collected one week later, and was euthanized. Serum was obtained by centrifugation, and the equal amount of each group was used as a mixed pool. The neutralizing antibody titer was determined according to the "Pathogen Inspection Manual" (edited by the National Institute of Infectious Diseases). The results calculated from the 50% plaque reduction rate are shown in Table 59. Compared with deactivated Japanese encephalitis virus particles, GA-immobilized Japanese encephalitis virus particles had the same or higher neutralizing antibody potency.
[表59] [Table 59]
7.在加速試驗下的穩定性(抗原含量) 以最終蛋白質濃度成為8μg/mL的方式,利用類PBS溶液來稀釋GA固定化日本腦炎病毒粒子及去活化日本腦炎病毒粒子。以抗原含量為指標,評估了於25℃的保存穩定性。作為代表,利用0.01w/v%的戊二醛濃度於4℃反應3日後的GA固定化日本腦炎病毒粒子或去活化日本腦炎病毒粒子的結果顯示於表60。GA固定化日本腦炎病毒粒子,在25℃保存下維持了1個月。另一方面,去活化日本腦炎病毒粒子,在25℃保存下顯示穩定性降低。結果顯示,與去活化日本腦炎病毒粒子相比,GA固定化日本腦炎病毒粒子的穩定性有提升。7. Stability under accelerated test (antigen content) GA-immobilized Japanese encephalitis virions and deactivated Japanese encephalitis virions were diluted with a PBS-like solution so that the final protein concentration was 8 μg/mL. The storage stability at 25°C was evaluated using the antigen content as an index. As a representative example, the results of GA-immobilized Japanese encephalitis virions or deactivated Japanese encephalitis virions after reacting at 4° C. for 3 days with a glutaraldehyde concentration of 0.01 w/v% are shown in Table 60. GA immobilized Japanese encephalitis virions and kept them for 1 month at 25°C. On the other hand, the deactivated Japanese encephalitis virion showed decreased stability when stored at 25°C. The results showed that the stability of GA-immobilized JE virions was improved compared with deactivated JE virions.
[表60] 括弧內是將第0日的抗原含量當作100%時,抗原含量變化的比例(%)[Table 60] The brackets are the ratio of antigen content change when the antigen content on day 0 is taken as 100% (%)
8.在加速試驗下的穩定性(動態光散射) 以最終蛋白質濃度成為8μg/mL的方式,利用類PBS溶液來稀釋GA固定化日本腦炎病毒粒子及去活化日本腦炎病毒粒子。以藉由使用了Zetasizer Nano ZS的動態光散射法測得的液體中的平均粒徑為指標,評估了於25℃的保存穩定性。作為代表,利用0.01w/v%的戊二醛濃度於4℃反應3日後的GA固定化日本腦炎病毒粒子或去活化日本腦炎病毒粒子的結果顯示於表61。GA固定化日本腦炎病毒粒子,在25℃保存下維持了1個月。另一方面,去活化日本腦炎病毒粒子,在25℃保存下顯示出粒徑增加。由此結果顯示,與去活化日本腦炎病毒粒子相比,GA固定化日本腦炎病毒粒子的穩定性有提升。8. Stability under accelerated test (dynamic light scattering) GA-immobilized Japanese encephalitis virions and deactivated Japanese encephalitis virions were diluted with a PBS-like solution so that the final protein concentration was 8 μg/mL. The storage stability at 25° C. was evaluated using the average particle diameter in liquid measured by the dynamic light scattering method using Zetasizer Nano ZS as an index. Table 61 shows the results of GA-immobilized Japanese encephalitis virions or deactivated Japanese encephalitis virions after reacting at a concentration of 0.01w/v% glutaraldehyde at 4°C for 3 days as a representative example. GA immobilized Japanese encephalitis virions and kept them for 1 month at 25°C. On the other hand, inactivated Japanese encephalitis virions showed an increase in particle size when stored at 25°C. These results indicated that the stability of GA-immobilized Japanese encephalitis virions was improved compared with deactivated Japanese encephalitis virions.
[表61] 括弧內是將第0日的平均粒徑當作100%時,平均粒徑變化的比例(%)[Table 61] The ratio (%) of the change in the average particle size when the average particle size on the 0th day is taken as 100% is in parentheses
9.在4℃保存下的穩定性(免疫原性) 以最終蛋白質濃度成為8μg/mL的方式,利用類PBS溶液來稀釋GA固定化日本腦炎病毒粒子及去活化日本腦炎病毒粒子。以在小鼠的免疫原性(中和抗體力價)為指標,評估了在4℃的保存穩定性。作為代表,利用0.01w/v%的戊二醛濃度於4℃反應15個月後的GA固定化日本腦炎病毒粒子或去活化日本腦炎病毒粒子的結果顯示於表62。雖然在0個月與15個月後投予量並不同,但推測出GA固定化日本腦炎病毒粒子,在4℃保存下維持15個月。另一方面,推測出去活化日本腦炎病毒粒子,在4℃保存下保存穩定性降低。並且可推測出,與去活化日本腦炎病毒粒子相比,GA固定化日本腦炎病毒粒子的穩定性有提升。9. Stability under storage at 4°C (immunogenicity) GA-immobilized Japanese encephalitis virions and deactivated Japanese encephalitis virions were diluted with a PBS-like solution so that the final protein concentration was 8 μg/mL. The storage stability at 4°C was evaluated using the immunogenicity (neutralizing antibody titer) in mice as an index. Table 62 shows the results of GA-immobilized Japanese encephalitis virions or deactivated Japanese encephalitis virions after reacting at 4° C. for 15 months at a glutaraldehyde concentration of 0.01 w/v% as a representative example. Although the doses administered were different between 0 and 15 months later, it was presumed that GA immobilized Japanese encephalitis virions and maintained them for 15 months under storage at 4°C. On the other hand, deactivated Japanese encephalitis virions are presumed to lower storage stability at 4°C. Furthermore, it can be presumed that the stability of GA-immobilized Japanese encephalitis virion is improved compared with deactivated Japanese encephalitis virion.
[表62] [Table 62]
[實施例9] FA固定化日本腦炎病毒粒子的製備 1.福馬林處理(對病毒粒子進行固定化粒子結構的步驟) 對去活化日本腦炎病毒粒子,以最終濃度成為0.014~0.04v/v%(以甲醛換算為0.005~0.015w/v%)的方式添加福馬林(36~38w/v%甲醛水溶液),於25℃使之反應1週。反應結束後,利用類PBS溶液來透析反應液,藉由除去福馬林,獲得了固定化日本腦炎病毒粒子(以下有時記載為「FA固定化日本腦炎病毒粒子」)。獲得的FA固定化日本腦炎病毒粒子的放熱活性,是藉由刺激了人類PBMC時的發炎性細胞激素的產量的定量來評估。[Example 9] Preparation of FA-immobilized Japanese encephalitis virus particles 1. Formalin treatment (the step of immobilizing the particle structure of virus particles) To the deactivated Japanese encephalitis virus particles, formalin (36-38w/v% formaldehyde aqueous solution) was added so that the final concentration became 0.014-0.04v/v% (0.005-0.015w/v% in formaldehyde conversion), and the It was reacted at 25°C for 1 week. After the reaction, the reaction solution was dialyzed against a PBS-like solution to remove formalin, thereby obtaining immobilized Japanese encephalitis virus particles (hereinafter sometimes referred to as "FA-immobilized Japanese encephalitis virus particles"). The exothermic activity of the obtained FA-immobilized JE virions was assessed by quantifying the production of inflammatory cytokines when human PBMCs were stimulated.
2.定量發炎性細胞激素的產量 以遵照歐洲藥典MONOCYTE-ACTIVATION TEST的方法,定量了利用FA固定化日本腦炎病毒粒子或去活化日本腦炎病毒粒子刺激了人類PBMC時的細胞激素(IL-1β及IL-6)的產量。具體而言,歐洲藥典MONOCYTE-ACTIVATION TEST中的將人類PBMC以最少4位供給者份作為混合池來使用的部份,變更成1位供給者份,而進行了測定。其結果顯示於表63。結果可知,與去活化日本腦炎病毒粒子相比,FA固定化日本腦炎病毒粒子的發炎性細胞激素的產量相當低。由此亦教示了,與去活化日本腦炎病毒粒子相比,FA固定化日本腦炎病毒粒子具有較高安全性。2. Quantification of Inflammatory Cytokines Production The production of cytokines (IL-1β and IL-6) was quantified when human PBMCs were stimulated with FA-immobilized Japanese encephalitis virions or deactivated Japanese encephalitis virions by following the method of the European Pharmacopoeia MONOCYTE-ACTIVATION TEST. Specifically, in the European Pharmacopoeia MONOCYTE-ACTIVATION TEST, human PBMC was used as a mixed pool with a minimum of 4 donors, and the measurement was performed by changing it to 1 donor. The results are shown in Table 63. As a result, it was found that the production of inflammatory cytokines in FA-immobilized Japanese encephalitis virions was considerably lower than that of deactivated Japanese encephalitis virions. This also teaches that FA-immobilized Japanese encephalitis virions are safer than deactivated Japanese encephalitis virions.
[表63] [Table 63]
[實施例10] 物性評估 用以下的方法評估了在上述實施例9所獲得的FA固定化日本腦炎病毒粒子的物性。[Example 10] Physical property evaluation The physical properties of the FA-immobilized Japanese encephalitis virus particles obtained in Example 9 above were evaluated by the following methods.
1.藉由電子顯微鏡實行的解析 為了詳細調查FA固定化日本腦炎病毒粒子的形狀,用與實施例2同樣的方法,實施了藉由電子顯微鏡的觀察。作為代表,是顯示以0.014v/v%的福馬林濃度於25℃反應1週後的FA固定化日本腦炎病毒粒子所拍攝成的相片(第5圖)。FA固定化日本腦炎病毒粒子,因為利用福馬林固定,所以與去活化日本腦炎病毒粒子同樣地保有粒子結構。1. Analysis by electron microscope In order to examine the shape of FA-immobilized Japanese encephalitis virus particles in detail, observation with an electron microscope was carried out in the same manner as in Example 2. As a representative, it is a photograph showing FA-immobilized Japanese encephalitis virions after reacting at 25°C for 1 week at a formalin concentration of 0.014v/v% (Fig. 5). FA-immobilized Japanese encephalitis virions retain their particle structure similarly to deactivated Japanese encephalitis virions because they are immobilized with formalin.
2.動態光散射 使用Zetasizer Nano ZS(Malvern公司製造)來解析FA固定化日本腦炎病毒粒子的平均粒徑。藉由動態光散射法測得的液體中的平均粒徑顯示於表64。FA固定化日本腦炎病毒粒子,是平均粒徑為約90nm且為單峰性。另一方面,去活化日本腦炎病毒粒子,是約80nm且為單峰性。又,FA固定化日本腦炎病毒粒子的粒子結構保持著,沒有確認到如凝集體般的雜質。2. Dynamic Light Scattering The average particle diameter of FA-immobilized Japanese encephalitis virus particles was analyzed using Zetasizer Nano ZS (manufactured by Malvern). The average particle size in the liquid measured by the dynamic light scattering method is shown in Table 64. The FA-immobilized Japanese encephalitis virus particles had an average particle diameter of about 90 nm and were unimodal. On the other hand, the inactivated Japanese encephalitis virions were about 80 nm and unimodal. Also, the particle structure of FA-immobilized Japanese encephalitis virus particles was maintained, and impurities such as aggregates were not confirmed.
[表64] [Table 64]
3.分子量分布測定(SEC) 用與實施例8同樣的方法(SEC)測定了FA固定化日本腦炎病毒粒子的分子量分布。溶出樣式顯示於表65。FA固定化日本腦炎病毒粒子是在溶出時間14~15分鐘附近確認到單峰性的主峰。又,去活化日本腦炎病毒粒子亦在溶出時間14~15分鐘附近確認到單峰性的主峰。3. Molecular Weight Distribution Determination (SEC) The molecular weight distribution of FA-immobilized Japanese encephalitis virus particles was measured by the same method (SEC) as in Example 8. The dissolution profile is shown in Table 65. In the FA-immobilized Japanese encephalitis virus particles, a unimodal main peak was confirmed around the elution time of 14 to 15 minutes. Also, in the case of deactivated Japanese encephalitis virus particles, a unimodal main peak was confirmed around the elution time of 14 to 15 minutes.
[表65] [Table 65]
4.抗原的含量 用與實施例8同樣的方法,藉由使用了抗日本腦炎病毒抗體的三明治ELISA法測定了抗原的含量(抗原含量)。4. Antigen content In the same manner as in Example 8, the antigen content (antigen content) was measured by the sandwich ELISA method using the anti-Japanese encephalitis virus antibody.
FA固定化日本腦炎病毒粒子及去活化日本腦炎病毒粒子各自的抗原含量顯示於表66。作為代表,將以0.014v/v%及0.02v/v%的福馬林濃度於25℃反應1週後的FA固定化日本腦炎病毒粒子進行評估後,其是與去活化日本腦炎病毒粒子包含了同等的抗原。Table 66 shows the respective antigen contents of FA-immobilized Japanese encephalitis virions and deactivated Japanese encephalitis virions. As a representative, FA-immobilized Japanese encephalitis virions after reacting at 25°C for 1 week at formalin concentrations of 0.014v/v% and 0.02v/v% were evaluated, which was consistent with deactivated Japanese encephalitis virions Contains equivalent antigens.
[表66] [Table 66]
5.免疫原性(小鼠腹腔內接種) 用與實施例8同樣的方法,作為代表,測定了以0.02v/v%的福馬林濃度於25℃反應1週後的FA固定化日本腦炎病毒粒子的中和抗體力價。由50%溶斑減少率算出的結果顯示於表67。與去活化日本腦炎病毒粒子相比,FA固定化日本腦炎病毒粒子為同等或在其以上的中和抗體力價。5. Immunogenicity (intraperitoneal inoculation of mice) Using the same method as in Example 8, as a representative, the neutralizing antibody titer of FA-immobilized Japanese encephalitis virions after reacting at 25°C for 1 week at a formalin concentration of 0.02v/v% was measured. The results calculated from the 50% plaque reduction rate are shown in Table 67. Compared with deactivated Japanese encephalitis virus particles, FA-immobilized Japanese encephalitis virus particles had the same or higher neutralizing antibody potency.
[表67] [Table 67]
6.在嚴格試驗下的穩定性(抗原含量) 以最終蛋白質濃度成為8μg/mL的方式,利用類PBS溶液來稀釋FA固定化日本腦炎病毒粒子及去活化日本腦炎病毒粒子。以抗原含量為指標,評估了於37℃的保存穩定性。作為代表,以0.014v/v%的福馬林濃度於25℃反應1週後的結果顯示於表68。FA固定化日本腦炎病毒粒子,在37℃保存下維持了1週。另一方面,去活化日本腦炎病毒粒子,在37℃保存下顯示穩定性降低。結果顯示出,與去活化日本腦炎病毒粒子相比,FA固定化日本腦炎病毒粒子的穩定性有提升。6. Stability under strict test (antigen content) FA-immobilized Japanese encephalitis virions and deactivated Japanese encephalitis virions were diluted with a PBS-like solution so that the final protein concentration was 8 μg/mL. The storage stability at 37°C was evaluated using the antigen content as an index. As a representative, the results after reacting at 25°C for 1 week at a formalin concentration of 0.014v/v% are shown in Table 68. FA-immobilized Japanese encephalitis virus particles were maintained at 37°C for 1 week. On the other hand, the deactivated Japanese encephalitis virion showed decreased stability when stored at 37°C. The results showed that the stability of FA-immobilized JE virions was improved compared to deactivated JE virions.
[表68]
〔實施例11〕 [Example 11]
對去活化日本腦炎病毒粒子,以最終濃度成為0.15~15mM的方式添加EDC,於4℃使之反應2~20小時。當進行阻停處理時,進一步將甘胺酸作為阻停劑,以EDC的8倍量(莫耳質量比)添加到反應液。反應結束後,利用類PBS溶液來透析反應液,藉由去除EDC及甘胺酸,獲得了固定化日本腦炎病毒粒子(以下有時記載為「EDC固定化日本腦炎病毒粒子」)。藉由刺激了人類PBMC時的發炎性細胞激素的產量的定量,評估了獲得的EDC固定化日本腦炎病毒粒子的放熱活性。 To the deactivated Japanese encephalitis virus particles, EDC was added so that the final concentration would be 0.15 to 15 mM, and reacted at 4°C for 2 to 20 hours. When the blocking treatment is carried out, glycine is further added to the reaction solution in an amount 8 times (molar mass ratio) of EDC as a blocking agent. After the reaction, the reaction solution was dialyzed with a PBS-like solution to obtain immobilized Japanese encephalitis virus particles (hereinafter sometimes referred to as "EDC-immobilized Japanese encephalitis virus particles") by removing EDC and glycine. The exothermic activity of the obtained EDC-immobilized Japanese encephalitis virions was evaluated by quantifying the production of inflammatory cytokines when human PBMCs were stimulated.
以遵照歐洲藥典MONOCYTE-ACTIVATION TEST的方法,作為代表,定量了細胞激素(IL-1β及IL-6)的產量,該細胞激素是藉由以0.15mM或1.5mM的EDC濃度於4℃反應20小時後的EDC固定化日本腦炎病毒粒子或去活化日本腦炎病毒粒子刺激了人類PBMC時的細胞激素。具體而言,歐洲藥典MONOCYTE-ACTIVATION TEST中的將人類PBMC以最少4位供給者份作為混合池來使用的部份,變更成1位供給者份,而進行了測定。其結果顯示於表69。結果可知,與去活化日本腦炎病毒粒子相比,EDC固定化日本腦炎病毒粒子的發炎性細胞激素產量相當低。由此亦教示了,與去活化日本腦炎病毒粒子相比,EDC固定化日本腦炎病毒粒子具有高安全性。Taking the method according to the European Pharmacopoeia MONOCYTE-ACTIVATION TEST as a representative, the production of cytokines (IL-1β and IL-6) was quantified by reacting at 4°C for 20 Hours after EDC immobilized JE virions or deactivated JE virions stimulated human PBMCs with cytokines. Specifically, in the European Pharmacopoeia MONOCYTE-ACTIVATION TEST, human PBMC was used as a mixed pool with a minimum of 4 donors, and the measurement was performed by changing it to 1 donor. The results are shown in Table 69. As a result, it was found that the production of inflammatory cytokines in EDC-immobilized Japanese encephalitis virions was considerably lower than that of deactivated Japanese encephalitis virions. This also teaches that the EDC-immobilized Japanese encephalitis virion has a high safety compared with the deactivated Japanese encephalitis virion.
[表69] [Table 69]
[實施例12] 物性評估 用以下的方法評估了在上述實施例11所獲得的EDC固定化日本腦炎病毒粒子的物性。[Example 12] Physical property evaluation The physical properties of the EDC-immobilized Japanese encephalitis virus particles obtained in Example 11 above were evaluated by the following methods.
1.藉由電子顯微鏡實行的解析 為了詳細調查EDC固定化日本腦炎病毒粒子的形狀,用與實施例2同樣的方法,實施了藉由電子顯微鏡的觀察。作為代表,以1.5mM的EDC濃度於4℃反應20小時後的EDC固定化日本腦炎病毒粒子所拍攝成的相片顯示於第6圖。EDC固定化日本腦炎病毒粒子,因為利用EDC固定,所以與去活化日本腦炎病毒粒子同樣地保有粒子結構。1. Analysis by electron microscope In order to investigate the shape of EDC-immobilized Japanese encephalitis virions in detail, observation with an electron microscope was carried out in the same manner as in Example 2. As a representative, photographs taken of EDC-immobilized Japanese encephalitis virus particles after reacting at 4°C for 20 hours at an EDC concentration of 1.5 mM are shown in Fig. 6 . EDC immobilizes Japanese encephalitis virus particles, and since EDC is used for immobilization, the particle structure is maintained in the same manner as deactivated Japanese encephalitis virus particles.
2.動態光散射 使用Zetasizer Nano ZS(Malvern公司製造)來解析EDC固定化日本腦炎病毒粒子的平均粒徑。藉由動態光散射法測得的液體中的平均粒徑顯示於表70。EDC固定化日本腦炎病毒粒子,平均粒徑為約90nm且為單峰性。另一方面,去活化日本腦炎病毒粒子,為約80nm且為單峰性。又,EDC固定化日本腦炎病毒粒子的粒子結構保持著,沒有確認到如凝集體般的雜質。2. Dynamic Light Scattering The average particle diameter of EDC-immobilized Japanese encephalitis virus particles was analyzed using Zetasizer Nano ZS (manufactured by Malvern). The average particle size in the liquid measured by the dynamic light scattering method is shown in Table 70. EDC immobilized Japanese encephalitis virions with an average particle size of about 90 nm and unimodality. On the other hand, the inactivated Japanese encephalitis virion was about 80 nm and unimodal. Also, the particle structure of EDC-immobilized Japanese encephalitis virus particles was maintained, and impurities such as aggregates were not confirmed.
[表70] [Table 70]
3.分子量測定(SEC) 用與實施例8同樣的方法測定了EDC固定化日本腦炎病毒粒子的分子量分布。溶出樣式顯示於表71。EDC固定化日本腦炎病毒粒子是在溶出時間14~15分鐘附近確認到單峰性的主峰。又,去活化日本腦炎病毒粒子亦在溶出時間14~15分鐘附近確認到單峰性的主峰。3. Molecular weight determination (SEC) The molecular weight distribution of EDC-immobilized Japanese encephalitis virus particles was measured in the same manner as in Example 8. The dissolution profile is shown in Table 71. In the EDC-immobilized Japanese encephalitis virus particles, a unimodal main peak was confirmed around the elution time of 14 to 15 minutes. Also, in the case of deactivated Japanese encephalitis virus particles, a unimodal main peak was confirmed around the elution time of 14 to 15 minutes.
[表71] [Table 71]
4.抗原的含量 用與實施例8同樣的方法,藉由使用了抗日本腦炎病毒抗體的三明治ELISA法測定了抗原的含量(抗原含量)。作為代表,將以1.5mM的EDC濃度於4℃反應20小時後的EDC固定化日本腦炎病毒粒子及去活化日本腦炎病毒粒子各自的抗原含量顯示於表72。EDC固定化日本腦炎病毒粒子,包含了與去活化日本腦炎病毒粒子同等的抗原。4. Antigen content In the same manner as in Example 8, the antigen content (antigen content) was measured by the sandwich ELISA method using the anti-Japanese encephalitis virus antibody. Table 72 shows the respective antigen contents of EDC-immobilized Japanese encephalitis virions and deactivated Japanese encephalitis virions after reacting at an EDC concentration of 1.5 mM at 4° C. for 20 hours as a representative example. EDC-immobilized Japanese encephalitis virions contain the same antigens as deactivated Japanese encephalitis virions.
[表72] [Table 72]
5.免疫原性(小鼠腹腔內接種) 用與實施例8同樣的方法,測定了EDC固定化日本腦炎病毒粒子的中和抗體力價。由50%溶斑減少率算出的結果顯示於表73。作為代表,以1.5mM的EDC濃度於4℃反應2小時後,將甘胺酸作為阻停劑,以EDC的8倍量(莫耳質量比)添加到反應液,將如此處理而成的EDC固定化日本腦炎病毒粒子進行評估後,其是與去活化日本腦炎病毒粒子相比為同等或在其以上的中和抗體力價。5. Immunogenicity (intraperitoneal inoculation of mice) In the same manner as in Example 8, the neutralizing antibody titer of EDC-immobilized Japanese encephalitis virus particles was measured. The results calculated from the 50% plaque reduction rate are shown in Table 73. As a representative, after reacting at 4°C for 2 hours at an EDC concentration of 1.5mM, glycine was used as a blocking agent, and 8 times the amount of EDC (molar mass ratio) was added to the reaction solution, and the EDC thus treated was The immobilized Japanese encephalitis virion was evaluated to have a neutralizing antibody potency equal to or higher than that of the deactivated Japanese encephalitis virion.
[表73] [Table 73]
6.穩定性(抗原含量) 以最終蛋白質濃度成為8μg/mL的方式,利用類PBS溶液來稀釋EDC固定化日本腦炎病毒粒子及去活化日本腦炎病毒粒子。以抗原含量為指標,評估了於25℃、37℃的保存穩定性。結果顯示於表74、表75。EDC固定化日本腦炎病毒粒子,在25℃保存下維持了1個月及在37℃保存下維持了1週。另一方面,去活化日本腦炎病毒粒子,無論是在25℃保存下、37℃保存下的任一者,均顯示出抗原含量的降低。結果顯示出,與去活化日本腦炎病毒粒子相比,EDC固定化日本腦炎病毒粒子的穩定性有提升。6. Stability (antigen content) EDC-immobilized Japanese encephalitis virions and deactivated Japanese encephalitis virions were diluted with a PBS-like solution so that the final protein concentration was 8 μg/mL. Using the antigen content as an index, the storage stability at 25°C and 37°C was evaluated. The results are shown in Table 74, Table 75. EDC immobilized Japanese encephalitis virus particles and maintained them for 1 month at 25°C and 1 week at 37°C. On the other hand, the inactivated Japanese encephalitis virions showed a decrease in the antigen content regardless of whether they were stored at 25°C or 37°C. The results showed that the stability of EDC-immobilized Japanese encephalitis virions was improved compared with deactivated Japanese encephalitis virions.
[表74] 括弧內是將第0日的抗原含量當作100%時,抗原含量變化的比例(%)[Table 74] The brackets are the ratio of antigen content change when the antigen content on day 0 is taken as 100% (%)
[表75] 括弧內是將第0日的抗原含量當作100%時,抗原含量變化的比例(%) [產業利用性][Table 75] The ratio (%) of the change in antigen content when the antigen content on day 0 is taken as 100% is in parentheses [Industrial Applicability]
本發明在醫藥品的領域,尤其是疫苗的領域中為有用的。The present invention is useful in the field of pharmaceuticals, especially in the field of vaccines.
無none
第1圖是利用電子顯微鏡將固定化流行性感冒病毒粒子拍攝而成的相片(福馬林處理)。 第2圖是利用電子顯微鏡將固定化流行性感冒病毒粒子拍攝而成的相片(戊二醛處理)。 第3圖是利用電子顯微鏡將固定化流行性感冒病毒粒子拍攝而成的相片(EDC處理)。 第4圖是利用電子顯微鏡將固定化日本腦炎病毒粒子拍攝而成的相片(戊二醛處理)。 第5圖是利用電子顯微鏡將固定化日本腦炎病毒粒子拍攝而成的相片(福馬林處理)。 第6圖是利用電子顯微鏡將固定化日本腦炎病毒粒子拍攝而成的相片(EDC處理)。Figure 1 is a photograph (formalin-treated) of immobilized influenza virus particles taken with an electron microscope. Figure 2 is a photograph (glutaraldehyde treatment) of immobilized influenza virus particles taken with an electron microscope. Fig. 3 is a photograph (EDC processing) of immobilized influenza virus particles taken with an electron microscope. Fig. 4 is a photograph (glutaraldehyde treatment) of immobilized Japanese encephalitis virus particles taken with an electron microscope. Fig. 5 is a photograph (formalin-treated) of immobilized Japanese encephalitis virus particles taken with an electron microscope. Fig. 6 is a photograph (EDC processing) of immobilized Japanese encephalitis virus particles taken with an electron microscope.
國內寄存資訊(請依寄存機構、日期、號碼順序註記) 無 國外寄存資訊(請依寄存國家、機構、日期、號碼順序註記) 無Domestic deposit information (please note in order of depositor, date, and number) none Overseas storage information (please note in order of storage country, institution, date, and number) none
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期刊 Delrue I, Verzele D, Madder A, Nauwynck HJ. Inactivated virus vaccines from chemistry to prophylaxis: merits, risks and challenges. Expert Rev Vaccines. 2012 Jun; 11(6): 695-719.; * |
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US10881723B2 (en) | 2021-01-05 |
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EP3403671A4 (en) | 2020-04-22 |
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